Aminooxy-modified nucleosidic compounds and oligomeric compounds prepared therefrom

ABSTRACT

Nucleosidic monomers and oligomeric compounds prepared therefrom are provided which have increased nuclease resistance, substituent groups (such as 2′-aminooxy groups) for increasing binding affinity to complementary strand, and regions of 2′-deoxy-erythro-pentofuranosyl nucleotides that activate RNase H. Such oligomeric compounds are useful for diagnostics and other research purposes, for modulating the expression of a protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to oligonucleotide therapeutics.

RELATED APPLICATION DATA

This patent application is a continuation-in-part of application Ser.No. 09/130,973 filed on Aug. 7, 1998, and application Ser. No.09/344,260 filed on Jun. 25, 1999, which is a continuation-in-part ofapplication Ser. No. 09/016,520, filed on Jan. 30, 1998, which claimspriority benefit of U.S. Provisional Application Ser. No. 60/037,143,filed on Feb. 14, 1997. The contents of each of the foregoingapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention is directed to aminooxy-modified nucleosides andoligonucleotides, to oligonucleotides that elicit RNase H for cleavagein a complementary nucleic acid strand, and to oligonucleotides whereinat least some of the nucleotides are functionalized to be nucleaseresistant, at least some of the nucleotides of the oligonucleotideincluding a substituent that potentiates hybridization of theoligonucleotide to a complementary strand of nucleic acid, and at leastsome of the nucleotides of the oligonucleotide include2′-deoxy-erythro-pentofuranosyl sugar moiety. The inclusion of one ormore aminooxy moieties in such oligonucleotide provides, inter alia, forimproved binding of the oligonucleotides to a complementary strand. Theoligonucleotides and macromolecules are useful for therapeutics,diagnostics and as research reagents.

BACKGROUND OF THE INVENTION

Oligonucleotides are known to hybridize to single-stranded RNA orsingle-stranded DNA. Hybridization is the sequence specific base pairhydrogen bonding of bases of the oligonucleotides to bases of target RNAor DNA. Such base pairs are said to be complementary to one another.

In determining the extent of hybridization of an oligonucleotide to acomplementary nucleic acid, the relative ability of an oligonucleotideto bind to the complementary nucleic acid may be compared by determiningthe melting temperature of a particular hybridization complex. Themelting temperature (T_(m)), a characteristic physical property ofdouble helices, denotes the temperature in degrees centigrade, at which50% helical (hybridized) versus coil (unhybridized) forms are present.T_(m) is measured by using the UV spectrum to determine the formationand breakdown (melting) of the hybridization complex. Base stackingwhich occurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Oligonucleotides can be used to effect enzymatic cleavage of a targetRNA by using the intracellular enzyme, RNase H. The mechanism of suchRNase H cleavage requires that a 2′-deoxyribofuranosyl oligonucleotidehybridize to a target RNA. The resulting DNA-RNA duplex activates theRNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavageof the RNA strand destroys the normal function of the RNA.Phosphorothioate oligonucleotides operate via this type of mechanism.However, for a DNA oligonucleotide to be useful for cellular activationof RNase H, the oligonucleotide must be reasonably stable to nucleasesin order to survive in a cell for a time period sufficient for RNase Hactivation. For non-cellular uses, such as use of oligonucleotides asresearch reagents, such nuclease stability may not be necessary.

Several publications of Walder et al. describe the interaction of RNaseH and oligonucleotides. Of particular interest are: (1) Dagle et al.,Nucleic Acids Research 1990, 18, 4751; (2) Dagle et al., AntisenseResearch And Development 1991, 1, 11; (3) Eder et al., J. Biol. Chem.1991, 266, 6472; and (4) Dagle et al., Nucleic Acids Research 1991, 19,1805. According to these publications, DNA oligonucleotides having bothunmodified phosphodiester internucleoside linkages and modifiedphosphorothioate internucleoside linkages are substrates for cellularRNase H. Since they are substrates, they activate the cleavage of targetRNA by RNase H. However, the authors further note that in Xenopusembryos, both phosphodiester linkages and phosphorothioate linkages arealso subject to exonuclease degradation. Such nuclease degradation isdetrimental since it rapidly depletes the oligonucleotide available forRNase H activation.

As described in references (1), (2) and (4), to stabilizeoligonucleotides against nuclease degradation while still providing forRNase H activation, 2′-deoxy oligonucleotides having a short section ofphosphodiester linked nucleotides positioned between sections ofphosphoramidate, alkyl phosphonate or phosphotriester linkages wereconstructed. Although the phosphoramidate-containing oligonucleotideswere stabilized against exonucleases, in reference (4) the authors notedthat each phosphoramidate linkage resulted in a loss of 1.6° C. in themeasured T_(m) value of the phosphoramidate containing oligonucleotides.Such a decrease in the T_(m) value is indicative of an decrease inhybridization between the oligonucleotide and its target strand.

Other authors have commented on the effect such a loss of hybridizationbetween an oligonucleotide and its target strand can have.Saison-Behmoaras et al., EMBO Journal 1991, 10, 1111, observed that eventhough an oligonucleotide could be a substrate for RNase H, cleavageefficiency by RNase H was low because of weak hybridization to the mRNA.The authors also noted that the inclusion of

an acridine substitution at the 3′ end of the oligonucleotide protectedthe oligonucleotide from exonucleases.

U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligomerscomprising an RNA oligomer, or a derivative thereof, conjugated to a DNAoligomer via a phosphodiester linkage. The RNA oligomers also bear2′-O-alkyl substituents. However, being phosphodiesters, the oligomersare susceptible to nuclease cleavage.

European Patent application 339,842, filed Apr. 13, 1989, discloses2′-O-substituted phosphorothioate oligonucleotides, including2′-O-methylribooligonucleotide phosphorothioate derivatives. Theabove-mentioned application also discloses 2′-O-methyl phosphodiesteroligonucleotides which lack nuclease resistance.

U.S. Pat. No. 5,149,797, issued Sep. 22, 1992, discloses mixed phosphatebackbone oligonucleotides which include an internal portion ofdeoxynucleotides linked by phosphodiester linkages, and flanked on eachside by a portion of modified DNA or RNA sequences. The flankingsequences include methyl phosphonate, phosphoromorpholidate,phosphoropiperazidate or phosphoramidate linkages.

U.S. Pat. No. 5,256,775, issued Oct. 26, 1993, describe mixedoligonucleotides that incorporate phosphoramidate linkages andphosphorothioate or phosphorodithioate linkages.

Although it has been recognized that cleavage of a target RNA strandusing an oligonucleotide and RNase H would be useful, nucleaseresistance of the oligonucleotide and fidelity of hybridization are ofgreat importance in the development of oligonucleotide therapeutics.Accordingly, there remains a long-felt need for methods and materialsthat could activate RNase H while concurrently maintaining or improvinghybridization properties and providing nuclease resistance. Sucholigonucleotides are also desired as research reagents and diagnosticagents.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with one embodiment of this invention there are providedcompounds of the structure:

wherein:

T₄ is Bx or Bx—L where Bx is a heterocyclic base moiety;

one of T₁, T₂ and T₃ is L, hydrogen, hydroxyl, a protected hydroxyl or asugar substituent group;

another one of T₁, T₂ and T₃ is L, hydroxyl, a protected hydroxyl, aconnection to a solid support or an activated phosphorus group;

the remaining one of T₁, T₂ and T₃ is L, hydrogen, hydroxyl or a sugarsubstituent group provided that at least one of T₁, T₂, T₃ and T₄ is Lor Bx—L;

said group L having one of the formulas;

 wherein:

each m and mm is, independently, from 1 to 10;

y is from 1 to 10;

E is N(R₁)(R₂) or N═C(R₁)(R₂);

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl wheresaid acyl is an acid, amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group;

or R₃ and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O.

In some preferred embodiments, one of T₁, T₂ or T₃ is L. In furtherpreferred embodiments T₃ is L.

In further preferred embodiments L is —O—(CH₂)₂—O—N(R₁)(R₂). In anotherpreferred embodiment R₁ is H or C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyland R₂ is Cl₁-C₁₀ substituted alkyl, preferably wherein R₁ is C₁-C₁₀alkyl and/or R₂ is NH₃ ⁺ or N(R₃)(R₄) C₁-C₁₀ substituted alkyl. Inanother preferred embodiment R₁ and R₂ are both C₁-C₁₀ substitutedalkyl, with preferred substituents being independently, NH₃ ⁺ orN(R₃)(R₄).

In some preferred embodiments Bx is adenine, guanine, hypoxanthine,uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.

In some preferred embodiments R₁ and R₂ are joined in a ring structurethat can include at least one heteroatom selected from N and O, withpreferred ring structures being imidazole, piperidine, morpholine or asubstituted piperazine wherein the substituent is prefereably C₁-C₁₂alkyl.

In some preferred embodiments T₁ is a protected hydroxyl. In otherpreferred embodiments T₂ is an activated phosphorus group or aconnection to a solid support. In some preferred embodiments, the solidsupport is microparticles. In further preferred embodiments the solidsupport material is CPG.

In some preferred embodiments L is bound to an exocyclic aminofunctionality of Bx. In other preferred embodiments, L is bound to acyclic carbon atom of Bx.

In further preferred embodiments T₄ is Bx—L. In still further preferredembodiments, Bx is adenine, 2-aminoadenine or guanine. In furtherpreferred embodiments Bx is a pyrimidine heterocyclic base and L iscovalently bound to C5 of Bx. In still further preferred embodiments Bxis a pyrimidine heterocyclic base and L is covalently bound to C4 of Bx.In yet further preferred embodiments Bx is a purine heterocyclic baseand L is covalently bound to N2 of Bx. In still further preferredembodiments Bx is a purine heterocyclic base and L is covalently boundto N6 of Bx.

In accordance with some preferred embodiments, there are providedoligomeric compounds which incorporate at least one nucleosidic compoundthat is functionalized to increase nuclease resistance of the oligomericcompounds. In a further embodiment oligomeric compounds arefunctionalized with a substituent group to increase their bindingaffinity to target RNAs.

The oligomeric compounds preferably comprise a plurality of nucleosideunits of the structure:

wherein:

T₄ of each nucleoside unit is, independently, Bx or Bx—L where Bx is aheterocyclic base moiety;

one of T₅, T₆ and T₇ of each nucleoside unit is, independently, L,hydroxyl, a protected hydroxyl, a sugar substituent group, an activatedphosphorus group, a connection to a solid support, a nucleoside, anucleotide, an oligonucleoside or an oligonucleotide;

another of T₅, T₆ and T₇ of each nucleoside unit is, independently, anucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;

the remaining one of T₅, T₆ and T₇ of each nucleoside unit is,independently, is L, hydrogen, hydroxyl, a protected hydroxyl, or asugar substituent group;

provided that on at least one of said nucleoside units T₄ is Bx—L or atleast one of T₅, T₆ and T₇ is L;

said group L having one of the formulas;

 wherein:

each m and mm is, independently, from 1 to 10;

y is from 1 to 10;

E is N(R₁)(R₂) or N═C(R₁)(R₂)

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl wheresaid acyl is acid, amide or ester,

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group orwherein R₃ and R₄ are joined in a ring structure that optionallyincludes an additional heteroatom selected from N and O.

In some preferred embodiments of the oligomeric compounds of theinvention, at least one of T₁, T₂ or T₃ is L. In further preferredembodiment, at least one T₃ is L.

In further preferred embodiments of the oligomeric compounds of theinvention, at least one L is —O—(CH₂)₂—O—N(R₁)(R₂). In further preferredembodiments of the oligomeric compounds of the invention, R₁ is H orC₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl and R₂ is C₁-C₁₀ substitutedalkyl, preferably wherein R₁ is C₁-C₁₀ alkyl and/or R₂ is NH₃ ⁺ orN(R₃)(R₄) C₁-C₁₀ substituted alkyl. In still further preferredembodiments of the oligomeric compounds of the invention, R₁ and R₂ areboth C₁-C₁₀ substituted alkyl, with preferred substituents beingindependently, NH₃ ⁺ or N(R₃)(R₄)

In some preferred embodiments of the oligomeric compounds of theinvention, Bx is adenine, guanine, hypoxanthine, uracil, thymine,cytosine, 2-aminoadenine or 5-methylcytosine.

In some preferred embodiments of the oligomeric compounds of theinvention, R₁ and R₂ are joined in a ring structure that can include atleast one heteroatom selected from N and O, with preferred ringstructures being imidazole, piperidine, morpholine or a substitutedpiperazine wherein the substituent is preferably C₁-C₁₂ alkyl.

In some preferred embodiments of the oligomeric compounds of theinvention, T₁ is a protected hydroxyl. In other preferred embodiments ofthe oligomeric compounds of the invention, T₂ is an activated phosphorusgroup or a connection to a solid support. In some preferred embodimentsof the oligomeric compounds of the invention, the solid support ismicroparticles. In further preferred embodiments the solid supportmaterial is CPG.

In some preferred embodiments of the oligomeric compounds of theinvention, L is bound to an exocyclic amino functionality of Bx. Inother preferred embodiments of the oligomeric compounds of theinvention, L is bound to a cyclic carbon atom of Bx.

In further preferred embodiments of the oligomeric compounds of theinvention, T₄ is Bx—L. In still further preferred embodiments, Bx isadenine, 2-aminoadenine or guanine. In further preferred embodiments ofthe oligomeric compounds of the invention, Bx is a pyrimidineheterocyclic base and L is covalently bound to C5 of Bx. In stillfurther preferred embodiments of the oligomeric compounds of theinvention, Bx is a pyrimidine heterocyclic base and L is covalentlybound to C4 of Bx. In yet further preferred embodiments of theoligomeric compounds of the invention, Bx is a purine heterocyclic baseand L is covalently bound to N2 of Bx. In still further preferredembodiments of the oligomeric compounds of the invention, Bx is a purineheterocyclic base and L is covalently bound to N6 of Bx.

In some preferred embodiments of the oligomeric compounds of theinvention, the oligomeric compounds are from 5 to 50 nucleoside units inlength. In further preferred embodiments of the oligomeric compounds ofthe invention, the oligomeric compounds are from 8 to 30 nucleosideunits in length, with 15 to 25 nucleoside units in length being morepporeferred.

In some preferred embodiments, chimeric oligomeric compounds areprovided that are specifically hybridizable with DNA or RNA comprising asequence of linked nucleoside units. Preferably, the sequence is dividedinto a first region having linked nucleoside units and a second regionbeing composed of linked nucleoside units having 2′-deoxy sugarmoieties. The linked nucleoside units of at least one of the first orsecond regions are connected by phosphorothioate linkages and at leastone of the linked nucleoside units of the first region bears a group Lthat is covalently attached to the heterocyclic base or the 2′, 3′ or 5′position of the sugar moiety wherein the group L has one of theformulas:

where

each m and mm is, independently, from 1 to 10;

y is from 1 to 10;

E is N(R₁)(R₂) or N═C(R₁)(R₂);

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein thesubstitution is OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl where theacyl is an acid, amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group; and

or R₃ and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O.

In some preferred embodiments, the nucleoside units of the first andsecond regions are connected by phosphorothioate internucleosidelinkages. In further preferred embodiments, the nucleoside units of thefirst region are connected by phosphodiester internucleoside linkagesand the nucleoside units of the second region are connected byphosphorothioate internucleoside linkages. In still further preferredembodiments, the nucleoside units of the first region are connected byphosphorothioate internucleoside linkages and the nucleoside units ofthe second region are connected by phosphodiester internucleosidelinkages.

In some preferred embodiments, the second region has at least threenucleoside units. In further preferred embodiments, the second regionhas at least five nucleoside units.

In some preferred embodiments, the chimeric oligomeric compound has athird region having 2′-O-alkyl substituted nucleoside units, wherein thesecond region is positioned between the first and third regions. Infurther preferred embodiments, the nucleoside units of the first, secondand third regions are connected by phosphorothioate linkages. In furtherpreferred embodiments, the nucleoside units of the first and thirdregions are connected by phosphodiester linkages and the nucleosideunits of the second region are connected by phosphorothioate linkages.In another preferred embodiment, the nucleoside units of the first andthird regions are connected by phosphorothioate linkages and thenucleoside units of the second region are connected by phosphodiesterlinkages.

In some preferred embodiments, the second region has at least threenucleoside units. In further preferred embodiments, the second regionhas at least five nucleoside units.

In some preferred embodiments, at least one of the 2′-O-alkylsubstituted nucleoside units of the third region bears an L group.

The nucleotides forming oligonucleotides of the present invention can beconnected via phosphorus linkages. Preferred phosphorous linkagesinclude phosphodiester, phosphorothioate and phosphorodithioatelinkages, with phosphodiester and phosphorothioate linkages beingparticularly preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous objects and advantages of the present invention may bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIG. 1 shows a synthesis of certain intermediates of the invention.

FIG. 2 shows a synthesis of 5-methyluridine DMT-phosphoramidate having aprotected aminooxyethyl group at the 2′-O position.

FIG. 3 shows a synthesis of certain intermediates of the invention.

FIG. 4 shows a synthesis of adenosine DMT-phosphoramidate having aprotected aminooxyethoxy group at the 2′ position.

FIG. 5 shows a synthesis of certain intermediates of the invention.

FIG. 6 shows a synthesis of cytidine DMT-phosphoramidate having aprotected aminooxyethoxy group at the 2′ position.

FIG. 7 shows a synthesis of certain intermediates of the invention.

FIG. 8 shows a synthesis of guanidine DMT-phosphoramidate having aprotected aminooxyethoxy group at the 2′ position.

FIG. 9 shows a synthesis of some intermediates and monomers of theinvention.

FIG. 10 shows a linking of compounds of the invention to CPG.

FIG. 11 shows a synthesis of intermediates and monomers of theinvention.

FIG. 12 shows a synthesis of intermediates and monomers of theinvention.

FIG. 13 shows a graph of % full length oligonucleotide versus time inminutes pertaining to effects of nuclease action on oligonucleotides.

FIG. 14 shows a graph of % full length oligonucleotide versus time inminutes pertaining to effects of nuclease action on oligonucleotides.

FIG. 15 shows a graph of % full length oligonucleotide versus time inminutes pertaining to effects of nuclease action on oligonucleotides.

FIG. 16 shows a synthesis of intermediates and monomers of theinvention.

FIG. 17 shows a synthesis of intermediates and monomers of theinvention.

FIG. 18 shows a synthesis of intermediates and monomers of theinvention.

FIG. 19 shows a synthesis of intermediates and monomers of theinvention.

FIG. 20 shows a synthesis of intermediates and monomers of theinvention.

FIG. 21 shows a synthesis of intermediates and monomers of theinvention.

FIG. 22 shows a synthesis of intermediates and monomers of theinvention.

FIG. 23 shows a synthesis of intermediates and monomers of theinvention.

FIG. 24 shows a synthesis of intermediates and monomers of theinvention.

FIG. 25 shows a synthesis of intermediates and monomers of theinvention.

FIG. 26 shows a synthesis of intermediates and monomers of theinvention.

FIG. 27 shows a synthesis of intermediates and monomers of theinvention.

FIG. 28 shows a synthesis of intermediates and monomers of theinvention.

FIG. 29 shows a synthesis of intermediates and monomers of theinvention.

FIG. 30 shows a synthesis of intermediates and monomers of theinvention.

FIG. 31 shows a synthesis of intermediates and monomers of theinvention.

FIG. 32 shows a synthesis of intermediates and DMT phosphoramiditemonomers of the invention.

FIG. 33 shows a synthesis of intermediates and monomers of the inventionattached to CPG.

FIG. 34 shows a synthesis of intermediates and DMT phosphoramiditemonomers of the invention.

FIG. 35 shows a synthesis of intermediates and DMT phosphoramiditemonomers of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention presents modified nucleosidic monomers andoligomers prepared therefrom. The monomers each comprise a nucleosidehaving at least one modification which is at a 2′, 3′ or 5′-sugarposition or which can be at a heterocyclic base position. More than oneposition can be modified in either the nucleosidic monomers or oligomersof the invention. The oligomeric compounds of the invention are usefulfor identification or quantification of an RNA or DNA or for modulatingthe activity of an RNA or DNA molecule. The oligomeric compounds havinga modified nucleosidic monomer therein are preferably prepared to bespecifically hybridizable with a preselected nucleotide sequence of asingle-stranded or double-stranded target DNA or RNA molecule. It isgenerally desirable to select a sequence of DNA or RNA which is involvedin the production of a protein whose synthesis is ultimately to bemodulated or inhibited in its entirety or to select a sequence of RNA orDNA whose presence, absence or specific amount is to be determined in adiagnostic test.

The nucleosidic monomers (monomers) of the invention are prepared havingone or more aminooxy modifications. The sites for modification can bethe 2′, 3′ and/or 5′ positions on the sugar portion, and/or in theheterocyclic base moiety of the monomers. In preferred embodiments, thenucleosidic monomers are of the formula:

wherein:

T₄ is Bx or Bx—L where Bx is a heterocyclic base moiety;

one of T₁, T₂ and T₃ is L, hydrogen, hydroxyl, a protected hydroxyl or asugar substituent group;

another one of T₁, T₂ and T₃ is L, hydroxyl, a protected hydroxyl, aconnection to a solid support or an activated phosphorus group;

the remaining one of T₁, T₂ and T₃ is L, hydrogen, hydroxyl or a sugarsubstituent group provided that at least one of T₁, T₂, T₃ and T₄ is Lor Bx—L;

said group L having one of the formulas;

 wherein:

each m and mm is, independently, from 1 to 10;

y is from 1 to 10;

E is N(R₁)(R₂) or N═C(R₁)(R₂);

each R₁ and R₂ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₃, SR₃, NH₃ ⁺, N(R₃)(R₄), guanidino or acyl wheresaid acyl is an acid, amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group;

or R₃ and R₄ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O.

While not wishing to be bound by a specific theory, the design ofaminooxy-modified oligomeric compounds is focused on a number of factorsthat include: an electronegative atom at the 2′-connecting site, whichis believed to be necessary for C_(3′)-endo conformation viaO_(4′)—O_(2′) gauche effect (increase in binding affinity); gaucheeffect of the 2′-substituent —O—CH₂—CH₂—O— (increase in bindingaffinity/nuclease resistance); restricted motion around N—O bond, as incalichiamycin, which is believed to lead to conformational constraintsin side chain; lipophilicity of the modification (which relates toprotein binding/absorption); and fusogenic properties of aminooxy sidechains.

One of the factors believed to be related to the2′-O-dimethylaminooxyethyl (DMAOE) substituent is the potentialfusogenic property or “proton sponge hypothesis.” The nitrogen of theDMAOE is expected to have pKa between 4.5 and 5.0. Thus, it is believedthat this nitrogen probably will not be protonated outside the cell orin cell membranes, but is likely to be protonated inside the endosomeswhere pH is around 5.0. Such a protonation is expected to prevent theendosomal degradation of the oligonucleotide by lysosomal nucleaseshaving an acidic optimal pH. Such a “proton sponge” is expected to alterthe osmolarity of the endosomal vesicle. The accumulation of protonsbrought in by the endosomal ATPase is coupled to an influx of chlorideanions. Concentration of DMAOE oligonucleotide in the endosome shouldcause an increase in the ionic concentration within the endosome,resulting in osmotic swelling of the endosome. Moreover, DMAOEprotonation is believed to cause internal charge repulsion. Both ofthese effects are believed to cause endosomal fusion to release theoligonucleotide to the cytoplasm. Once the oligonucleotide is in thecytoplasm, it should be easily transported to the nucleus.

It is preferred that the oligonucleotides of the invention be adapted tobe specifically hybridizable with the nucleotide sequence of the targetRNA or DNA selected for modulation. Oligonucleotides particularly suitedfor the practice of one or more embodiments of the present inventioncomprise 2′, 3′, or 5′-sugar modified or heterocyclic base modifiedoligonucleotides wherein the modification is an aminooxy moiety. Forexample, the oligonucleotides are modified to contain substitutionsincluding but not limited incorporation of one or more nucleoside unitsmodified as shown in the formula defining “L” above. The modifiednucleosidic compounds can be positioned internally in theoligonucleotide via linking in the oligonucleotide backbone or they canbe located on one or both of the 3′ and 5′ terminal ends of theoligonucleotide.

The nucleosidic monomers of the present invention can includeappropriate activated phosphorus groups such as activated phosphategroups and activated phosphite groups. As used herein, the termsactivated phosphate and activated phosphite groups refer to activatedmonomers or oligomers that are reactive with a hydroxyl group of anothermonomeric or oligomeric compound to form a phosphorus-containinginternucleotide linkage. Such activated phosphorus groups containactivated phosphorus atoms in P^(III) or P^(V) valency states. Suchactivated phosphorus atoms are known in the art and include, but are notlimited to, phosphoramidite, H-phosphonate and phosphate triesters. Apreferred synthetic solid phase synthesis utilizes phosphoramidites asactivated phosphates. The phosphoramidites utilize P^(III) chemistry.The intermediate phosphite compounds are subsequently oxidized to theP^(V) state using known methods to yield, in preferred embodiments,phosphodiester or phosphorothioate internucleotide linkages. Additionalactivated phosphates and phosphites are disclosed in Tetrahedron ReportNumber 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

The oligomers (oligomeric compounds) of the invention are convenientlysynthesized using solid phase synthesis of known methodology, and arepreferably designed to be complementary to or specifically hybridizablewith a preselected nucleotide sequence of the target RNA or DNA.Standard solution phase and solid phase methods for the synthesis ofoligonucleotides and oligonucleotide analogs are well known to thoseskilled in the art. These methods are constantly being improved in waysthat reduce the time and cost required to synthesize these complicatedcompounds. Representative solution phase techniques are described inU.S. Pat. No. 5,210,264, issued May 11, 1993 and commonly assigned withthis invention. Representative solid phase techniques employed foroligonucleotide and oligonucleotide analog synthesis utilizing standardphosphoramidite chemistries are described in, Protocols ForOligonucleotides And Analogs, Agrawal, S., ed., Humana Press, Totowa,N.J., 1993.

The oligomeric compounds of the invention also include those thatcomprise nucleosides connected by charged linkages, and whose sequencesare divided into at least two regions. In some preferred embodiments,the first region includes 2′-aminooxyalkyl substituted-nucleosideslinked by a first type of linkage, and the second region includesnucleosides linked by a second type of linkage. In seom preferredembodiments, the oligomers of the invention further include a thirdregion comprised of nucleosides as are used in the first region, withthe second region positioned between the first and the third regions.Such oligomeric compounds are known as “chimeras,” “chimeric,” or“gapped” oligonucleotides. (See, e.g., U.S. Pat. No. 5,623,065, issuedApr. 22, 1997, the contents of which are incorporated herein byreference.)

GAPmer technology has been developed to incorporate modifications at theends (“wings”) of oligomeric compounds, leaving a phosphorothioate Gapin the middle for RNase H activation (Cook, P. D., Anti-Cancer DrugDes., 1991, 6, 585-607; Monia et al., J. Biol. Chem ., 1993, 268,14514-14522). In a recent report, the activities of a series ofuniformly 2′-O modified 20 mer RNase H-independent oligonucleotides thatwere antisense to the 5′-cap region of human ICAM-1 transcript in HUVECcells, were compared to the parent 2′-deoxy phosphorothioateoligonucleotide. See Baker et al., J. Bio. Chem., 1997, 272,11994-12000). The 2′-MOE/P═O oligomer demonstrated the greatest activitywith a IC₅₀ of 2.1 nM (T_(m)=87.1° C.), while the parent P═Soligonucleotide analog had an IC₅₀ of 6.5 nM (T_(m)=79.2° C.)Correlation of activity with binding affinity was not always seen as the2′-F/P═S (T_(m)=87.9° C.) was less active than the 2′-MOE/P═S(T_(m)=79.2° C.) by four fold. The RNase H competent 2′-deoxy P═S parentoligonucleotide exhibited an IC₅₀=41 nM.

In the context of this invention, the terms “oligomer” and “oligomericcompound” refer to a plurality of naturally occurring or non-naturallyoccurring nucleosides joined together in a specific sequence. “Oligomer”and “oligomeric compound” include oligonucleotides, oligonucleotideanalogs and chimeric oligomeric compounds having non-phosphoruscontaining internucleoside linkages. In some preferred embodiments, eachof the oligomeric compounds of the invention have at least one modifiednucleoside where the modification is an aminooxy compound of theinvention. Preferred nucleosides of the invention are joined through asugar moiety via phosphorus linkages, and include those containingadenine, guanine, adenine, cytosine, uracil, thymine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, other aza anddeaza thymidines, other aza and deaza cytosines, other aza and deazaadenines, other aza and deaza guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Heterocyclic base moieties (often referred to in the art simply as the“base”) amenable to the present invention include both naturally andnon-naturally occurring nucleobases and heterocycles. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenineand guanine, and the pyrimidine bases thymine, cytosine and uracil.Modified nucleobases include other synthetic and natural nucleobasessuch as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No.08/762,587, now U.S. Pat.No. 5,808,027 filed on Dec. 10, 1996, also herein incorporated byreference.

The preferred sugar moieties are deoxyribose or ribose. However, othersugar substitutes known in the art are also amenable to the presentinvention.

As used herein, the term “sugar substituent groups” refer to groups thatare attached to sugar moieties of compounds or oligomers of theinvention. Sugar substituent groups are covalently attached at sugar 2′,3′ and 5′-positions. In some preferred embodiments, the sugarsubstituent group has an oxygen atom bound directly to the 2′, 3′ and/or5′-carbon atom of the sugar. Preferably, sugar substituent groups areattached at 2′-positions although sugar substituent groups may also belocated at 3′ and 5′ positions.

Sugar substituent groups amenable to the present invention includefluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino,O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula(O-alkyl)_(m), where m is 1 to about 10. Preferred among thesepolyethers are linear and cyclic polyethylene glycols (PEGs), and(PEG)-containing groups, such as crown ethers and those which aredisclosed by Ouchi, et al., Drug Design and Discovery 1992, 9, 93,Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardo et. al.,Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249.Further sugar modifications are disclosed in Cook, P. D., Anti-CancerDrug Design, 1991, 6, 585-607. Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. patent application Ser. No. 08/398,901, allowed , filed Mar. 6,1995, entitled Oligomeric Compounds having Pyrimidine Nucleotide(s) with2′ and 5′ Substitutions, hereby incorporated by reference in itsentirety.

Additional sugar substituent groups amenable to the present inventioninclude —SR and —NR₂ groups, where each R is, independently, hydrogen, aprotecting group or substituted or unsubstituted alkyl, alkenyl, oralkynyl. 2′-SR nucleosides are disclosed in U.S. Pat. No. 5,670,633,issued Sep. 23, 1997, hereby incorporated by reference in its entirety.The incorporation of 2′-SR monomer synthons are disclosed by Hamm etal., J. Org. Chem., 1997, 62, 3415-3420. 2′-NR₂ nucleosides aredisclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; andPolushin et al., Tetrahedron Lett., 1996, 37, 3227-3230.

Further representative sugar substituent groups amenable to the presentinvention include those having one of formula I or II:

wherein

Z₀ is O, S or NH;

E is C₁-C₁₀ alkyl, N(R₄)(R₅) or N═C(R₄)(R₅);

each R₄ and R₅ is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, whereinsaid substitution is OR₆, SR₆, NH₆ ⁺, N(R₆)(R₇), guanidino or acyl wheresaid acyl is an acid amide or an ester;

or R₄ and R₅, together, are a nitrogen protecting group or are joined ina ring structure that optionally includes an additional heteroatomselected from N and O; and

each R₆ and R₇ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protectinggroup, or R₃ and R₄, together, are a nitrogen protecting group;

or R₆ and R₇ are joined in a ring structure that optionally includes anadditional heteroatom selected from N and O;

R₃ is OX, SX, or N(X)₂;

each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)Z,C(═O)N(H)Z or OC(═O)N(H)Z;

Z is H or C₁-C₈ alkyl;

L₁, L₂ and L₃ comprise a ring system having from about 4 to about 7carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R₄)(R₅) OR₄, halo, SR₄or CN;

each q₁ is, independently, from 2 to 10;

each q₂ is 0 or 1;

p is from 1 to 10; and

r is from 1 to 10 with the proviso that when p is 0, r is greater than1.

Representative 2′-O- sugar substituents of formula I are disclosed inU.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998,entitled Capped 2′-Oxyethoxy Oligonucleotides, hereby incorporated byreference in its entirety.

Representative cyclic 2′-O- sugar substituents of formula II aredisclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27,1998, entitled RNA Targeted 2′-Modified Oligonucleotides that areConformationally Preorganized, hereby incorporated by reference in itsentirety.

Particularly preferred sugar substituent groups includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n NH) ₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10.

Some preferred oligomeric compounds of the invention contain at leastone nucleoside having one of the following at the 2′ position: C₁ to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF_(3,) SOCH₃, SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, an RNA cleaving group, a reportergroup, an intercalator, a group for improving the pharmacokineticproperties of an oligonucleotide, or a group for improving thepharmacodynamic properties of an oligonucleotide, and other substituentshaving similar properties. A preferred modification includes2′-methoxyethoxy [2′-O-CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), i.e., analkoxyalkoxy group. A further preferred modification is2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in co-owned U.S. patent application Ser. No.09/016,520, filed on Jan. 30, 1998, the contents of which are hereinincorporated by reference.

Other preferred modifications include 2′-methoxy (2′-O-CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/468,037, now U.S.Pat. No. 5,859,221 filed on Jun. 5, 1995, also herein incorporated byreference.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O include,but are not limited to, S, CH₂, CHF, and CF₂. See, e.g., Secrist et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992,

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′-position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of thepresent invention involves chemically linking to the oligonucleotide oneor more moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude, but are not limited to, lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053),a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264,229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety(Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Compounds of the invention can include ring structures that include anitrogen atom (e.g., —N(R₁)(R₂) and —N(R₃)(R₄) where (R₁)(R₂) and(R₃)(R₄) each form cyclic structures about the respective N) . Theresulting ring structure is a heterocycle or a heterocyclic ringstructure that can include further heteroatoms selected from N, O and S.Such ring structures may be mono-, bi- or tricyclic, and may besubstituted with substituents such as oxo, acyl, alkoxy, alkoxycarbonyl,alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl,carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy,hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, sulfone, sulfonamide,thiol and thioalkoxy. A preferred bicyclic ring structure that includesnitrogen is phthalimido.

Heterocyclic ring structures of the present invention can be fullysaturated, partially saturated, unsaturated or with a polycyclicheterocyclic ring each of the rings may be in any of the availablestates of saturation. Heterocyclic ring structures of the presentinvention also include heteroaryl which includes fused systems includingsystems where one or more of the fused rings contain no heteroatoms.Heterocycles, including nitrogen heterocycles, according to the presentinvention include, but are not limited to, imidazole, pyrrole, pyrazole,indole, 1H-indazole, α-carboline, carbazole, phenothiazine, phenoxazine,tetrazole, triazole, pyrrolidine, piperidine, piperazine and morpholinegroups. A more preferred group of nitrogen heterocycles includesimidazole, pyrrole, indole, and carbazole groups.

The present invention provides oligomeric compounds comprising aplurality of linked nucleosides wherein the preferred internucleosidelinkage is a 3′, 5′-linkage. Alternatively, 2′, 5′-linkages can be used(as described in U.S. application Ser. No. 09/115,043, filed Jul. 14,1998). A 2′, 5′-linkage is one that covalently connects the 2′-positionof the sugar portion of one nucleotide subunit with the 5′-position ofthe sugar portion of an adjacent nucleotide subunit.

The oligonucleotides of the present invention preferably are about 5 toabout 50 bases in length. It is more preferred that the oligonucleotidesof the invention have from 8 to about 30 bases, and even more preferredthat from about 15 to about 25 bases be employed.

In positioning one of the nucleosidic monomers of the invention in anoligonucleotide, an appropriate blocked and activated monomer isincorporated in the oligonucleotides in the standard manner forincorporation of a normal blocked and active standard nucleotide. forexample, a diisopropyl phosphoramidite nucleosidic monomer is selectedthat has an aminooxy moiety that is protected with, for example, aphthalimido protecting group. In addition, one of the hydroxyl groups ofthe nucleosidic monomer molecule, for example the 5′-hydroxyl, isprotected with a dimethoxytrityl (DMT) protecting group, and the otherhydroxyl group, (i.e., the 3′-hydroxyl group), bears a cyanoethylprotecting group. The nucleosidic monomer is added to the growingoligonucleotide by treating with the normal activating agents, as isknown is the art, to react the phosphoramidite moiety with the growingoligonucleotide. This is followed by removal of the DMT group in thestandard manner, as is known in the art, and continuation of elongationof the oligonucleotide with normal nucleotide amidite units as isstandard in the art. If the nucleosidic monomer is an intermediate unitutilized during synthesis of the oligonucleotide, the nucleosidicmonomer nucleoside is positioned in the interior of the oligonucleotide.If the nucleosidic monomer is the last unit linked to theoligonucleotide, the nucleosidic monomer will form the 5′ most terminalmoiety of the oligonucleotide. There are a plurality of alternativemethods for preparing oligomeric compounds of the invention that arewell known in the art. The phosphoramidite method illustrated above ismeant as illustrative of one of these methods.

In the context of this specification, alkyl (generally C₁-C₁₀), alkenyl(generally C₂-C₁₀), and alkynyl (generally C₂-C₁₀) groups include butare not limited to substituted and unsubstituted straight chain, branchchain, and alicyclic hydrocarbons, including methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl,nonadecyl, eicosyl and other higher carbon alkyl groups. Furtherexamples include 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylbutyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2-ethylhexyl and other branched chain groups, allyl, crotyl, propargyl,2-pentenyl and other unsaturated groups containing a pi bond,cyclohexane, cyclopentane, adamantane as well as other alicyclic groups,3-penten-2-one, 3-methyl-2-butanol, 2-cyanooctyl, 3-methoxy-4-heptanal,3-nitrobutyl, 4-isopropoxydodecyl, 4-azido-2-nitrodecyl,5-mercaptononyl, 4-amino-1-pentenyl as well as other substituted groups.

Further, in the context of this invention, a straight chain compoundmeans an open chain compound, such as an aliphatic compound, includingalkyl, alkenyl, or alkynyl compounds; lower alkyl, alkenyl, or alkynylas used herein include but are not limited to hydrocarbyl compounds fromabout 1 to about 6 carbon atoms. A branched compound, as used herein,comprises a straight chain compound, such as an alkyl, alkenyl, alkynylcompound, which has further straight or branched chains attached to thecarbon atoms of the straight chain. A cyclic compound, as used herein,refers to closed chain compounds, i.e. a ring of carbon atoms, such asan alicyclic or aromatic compound. The straight, branched, or cycliccompounds may be internally interrupted, as in alkoxy or heterocycliccompounds. In the context of this invention, internally interruptedmeans that the carbon chains may be interrupted with heteroatoms such asO, N, or S. However, if desired, the carbon chain may have noheteroatoms.

In one aspect of the invention the overall length of the alkyl groupappended to a nucleosidic monomer will be selected to be less than 11with the aminooxy group positioned between the ends of the alkyl group.In certain preferred nucleoside monomers of the invention, it ispreferred to position the aminooxy group with at least two methylenegroups between it and either of the hydroxyl groups of the nucleosidemonomer. This can be accomplished by any combination of methylene unitsin either the alkyl backbone or on the aminooxy side chain. As sopositioned the oxygen atom of the aminooxy moiety and the oxygen atomsof the hydroxyl groups do not form acetal type structures. In otherembodiments the aminooxy moiety is positioned with only one methylenegroup between it and one or the other of the hydroxyl groups forming anacetal type structure.

In substituted nucleosidic monomers of the invention, a first preferredgroup of substituents include 2′-O-aminoxyalkyl substituents. A furtherpreferred group of substituents includes 2′-O-alkylaminooxyalkyl,2′-O-di-alkylaminooxyalkyl and 2′-O-monoalkylaminooxyalkyl, e.g.,dimethylaminooxyethyl and ethylaminooxyethyl. An additional preferredgroup of substituents include precursor or blocked forms of these2′-O-aminooxyalkyl substituents include phthalimido and formaldehydeadducts, i.e., phthalimido-N-oxy and formaloximyl groups. A morepreferred group of substituents includes 2′-aminooxyalkyl where theamino group is substituted with one or more substituted alkyl groupswhere preferred substitutions are amino and substituted amino.

In certain preferred embodiments of the present invention, oligomericcompounds are linked via phosphorus linkages. Preferred phosphoruslinkages include phosphodiester, phosphorothioate and phosphorodithioatelinkages. In one preferred embodiment of this invention, nucleaseresistance is conferred on the oligonucleotides by utilizingphosphorothioate internucleoside linkages.

As used herein, the term oligonucleoside includes oligomers or polymerscontaining two or more nucleoside subunits having a non-phosphorouslinking moiety. Oligonucleosides according to the invention havemonomeric subunits or nucleosides having a ribofuranose moiety attachedto a heterocyclic base moiety through a glycosyl bond.

Oligonucleotides and oligonucleosides can be joined to give a chimericoligomeric compound. In addition to the naturally occurringphosphodiester linking group, phosphorus and non-phosphorus containinglinking groups that can be used to prepare oligonucleotides,oligonucleosides and oligomeric chimeric compounds (oligomericcompounds) of the invention are well documented in the prior art andinclude without limitation the following:

phosphorus containing linkaqes phosphorodithioate (—O—P(S)(S)—O—);

phosphorothioate (—O—P(S)(O)—O—);

phosphoramidate (—O—P(O)(NJ)—O—);

phosphonate (—O—P(J)(O)—O—);

phosphotriesters (—O—P(O J)(O)—O—);

phophosphoramidate (—O—P(O)(NJ)—S—);

thionoalkylphosphonate (—O—P(S)(J)—O—);

thionoalkylphosphotriester (—O—P(O)(OJ)—S—);

boranophosphate (—R⁵—P(O)(O)—J—);

non-phosphorus containing linkages

thiodiester (—O—C(O)—S—);

thionocarbamate (—O—C(O)(NJ)—S—);

siloxane (—O—Si(J)₂—O—);

carbamate (—O—C(O)—NH— and —NH—C(O)—O—)

sulfamate (—O—S(O)(O)—N— and —N—S(O)(O)—N—;

morpholino sulfamide (—O—S(O)(N(morpholino)—);

sulfonamide (—O—SO₂—NH—);

sulfide (—CH₂—S—CH₂—);

sulfonate (—O—SO₂—CH₂—)

N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—);

thioformacetal (—S—CH₂—O—);

formacetal (—O—CH₂—O—);

thioketal (—S—C(J)₂—O—); and

ketal (—O—C(J)₂—O—);

amine (—NH—CH₂—CH₂—)

hydroxylamine (—CH₂—N(J)—O—);

hydroxylimine (—CH═N—O—); and

hydrazinyl (—CH₂—N(H)—N(H)—).

“J” denotes a substituent group which is commonly hydrogen or an alkylgroup, but which can be a more complicated group that varies from onetype of linkage to another.

In addition to linking groups as described above that involve themodification or substitution of one or more of the —O—P(O)₂—O— atoms ofa naturally occurring linkage, included within the scope of the presentinvention are linking groups that include modification of the5′-methylene group as well as one or more of the atoms of the naturallyoccurring linkage. Linkages of this type are well documented in theliterature and include without limitation the following:

amides (—CH₂—CH₂—N(H)—C(O)) and —CH₂—O—N═CH—; and

alkylphosphorus (—C(J)₂—P(═O)(OJ)—C(J)₂—C(J)₂—).

wherein J is as described above.

Synthetic schemes for the synthesis of the substitute internucleosidelinkages described above are disclosed in: WO 91/08213; WO 90/15065; WO91/15500; WO 92/20822; WO 92/20823; WO 91/15500; WO 89/12060; EP 216860;U.S. Pat. Nos. 9,204,294; 9,003,138; 9,106,855; 9,203,385; 9,103,680;U.S. Pat. Ser. Nos. 07/990,848; 07,892,902; 07/806,710; 07/763,130;07/690,786; U.S. Pat. No. 5,466,677; 5,034,506; 5,124,047; 5,278,302;5,321,131; 5,519,126; 4,469,863; 5,455,233; 5,214,134; 5,470,967;5,434,257; Stirchak, E. P., et al., Nucleic Acid Res., 1989, 17,6129-6141; Hewitt, J. M., et al., 1992, 11, 1661-1666; Sood, A., et al.,J. Am. Chem. Soc., 1990, 112, 9000-9001; Vaseur, J. J. et al., J. Amer.Chem. Soc., 1992, 114, 4006-4007; Musichi, B., et al., J. Org. Chem.,1990, 55, 4231-4233; Reynolds, R. C., et al., J. Org. Chem., 1992, 57,2983-2985; Mertes, M. P., et al., J. Med. Chem., 1969, 12, 154-157;Mungall, W. S., et al., J. Org. Chem., 1977, 42, 703-706; Stirchak, E.P., et al.., J. Org. Chem., 1987, 52, 4202-4206; Coull, J. M., et al.,Tet. Lett., 1987, 28, 745; and Wang, H., et al., Tet. Lett., 1991, 32,7385-7388.

Other modifications can be made to the sugar, to the base, or to thephosphate group of the nucleotide. Representative modifications aredisclosed in International Publication Numbers WO 91/10671, publishedJul. 25, 1991, WO 92/02258, published Feb. 20, 1992, WO 92/03568,published Mar. 5, 1992, and U.S. Pat. Nos. 5,138,045, 5,218,105,5,223,618 5,359,044, 5,378,825, 5,386,023, 5,457,191, 5,459,255,5,489,677, 5,506,351, 5,541,307, 5,543,507, 5,571,902, 5,578,718,5,587,361, 5,587,469, all assigned to the assignee of this application.The disclosures of each of the above referenced U.S. patents are hereinincorporated by reference.

The attachment of conjugate groups to oligonucleotides and analogsthereof is well documented in the prior art. The compounds of theinvention can include conjugate groups covalently bound to functionalgroups such as primary or secondary hydroxyl groups. Conjugate groups ofthe invention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, phospholipids, biotin, phenazine, phenanthridine,anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.Groups that enhance the pharmacodynamic properties, in the context ofthis invention, include groups that improve oligomer uptake, enhanceoligomer resistance to degradation, and/or strengthen sequence-specifichybridization with RNA. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Representative conjugate groups are disclosed in International PatentApplication PCT/US92/09196, filed Oct. 23, 1992, U.S. Pat. No.5,578,718, issued Jul. 1, 1997, and U.S. Pat. No. 5,218,105. Each of theabove U.S. patents of the foregoing is commonly assigned with thisapplication. The entire disclosure of each U.S. patent is incorporatedherein by reference.

Other groups for modifying antisense properties include RNA cleavingcomplexes, pyrenes, metal chelators, porphyrins, alkylators, hybridintercalator/ligands and photo-crosslinking agents. RNA cleavers includeo-phenanthroline/Cu complexes and Ru(bipyridine)₃ ²⁺ complexes. TheRu(bpy)₃ ²⁺ complexes interact with nucleic acids and cleave nucleicacids photochemically. Metal chelators are include EDTA, DTPA, ando-phenanthroline. Alkylators include compounds such as iodoacetamide.Porphyrins include porphine, its substituted forms, and metal complexes.Pyrenes include pyrene and other pyrene-based carboxylic acids thatcould be conjugated using the similar protocols.

Hybrid intercalator/ligands include the photonuclease/intercalatorligand6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoyl-pentafluorophenylester. This compound has two noteworthy features: an acridine moietythat is an intercalator and a p-nitro benzamido group that is aphotonuclease.

Photo-crosslinking agents include aryl azides such as, for example,N-hydroxysucciniimidyl-4-azidobenzoate (HSAB) andN-succinimidyl-6(-4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH). Arylazides conjugated to oligonucleotides effect crosslinking with nucleicacids and proteins upon irradiation. They also crosslink with carrierproteins (such as KLH or BSA), raising antibody against theoligonucleotides.

Vitamins according to the invention generally can be classified as watersoluble or lipid soluble. Water soluble vitamins include thiamine,riboflavin, nicotinic acid or niacin, the vitamin B₆ pyridoxal group,pantothenic acid, biotin, folic acid, the B₁₂ cobamide coenzymes,inositol, choline and ascorbic acid. Lipid soluble vitamins include thevitamin A family, vitamin D, the vitamin E tocopherol family and vitaminK (and phytols). The vitamin A family, including retinoic acid andretinol, are absorbed and transported to target tissues through theirinteraction with specific proteins such as cytosol retinol-bindingprotein type II (CRBP-II), Retinol-binding protein (RBP), and cellularretinol-binding protein (CRBP). These proteins, which have been found invarious parts of the human body, have molecular weights of approximately15 kD. They have specific interactions with compounds of vitamin-Afamily, especially, retinoic acid and retinol.

In the context of this invention, “hybridization” shall mean hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleotides. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. “Complementary,” as used herein, alsorefers to sequence complementarity between two nucleotides. For example,if a nucleotide at a certain position of an oligonucleotide is capableof hydrogen bonding with a nucleotide at the same position of a DNA orRNA molecule, then the oligonucleotide and the DNA or RNA are consideredto be complementary to each other at that position. The oligonucleotideand the DNA or RNA are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between the oligonucleotide and the DNA or RNA target. Itis understood that an oligonucleotide need not be 100% complementary toits target DNA sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e. under physiological conditions in thecase of in vivo assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed.

Cleavage of oligonucleotides by nucleolytic enzymes requires theformation of an enzyme-substrate complex, or in particular, anuclease-oligonucleotide complex. The nuclease enzymes will generallyrequire specific binding sites located on the oligonucleotides forappropriate attachment. If the oligonucleotide binding sites are removedor blocked, such that nucleases are unable to attach to theoligonucleotides, the oligonucleotides will be nuclease resistant. Inthe case of restriction endonucleases that cleave sequence-specificpalindromic double-stranded DNA, certain binding sites such as the ringnitrogen in the 3- and 7-positions have been identified as requiredbinding sites. Removal of one or more of these sites or stericallyblocking approach of the nuclease to these particular positions withinthe oligonucleotide has provided various levels of resistance tospecific nucleases.

This invention provides oligonucleotides possessing superiorhybridization properties. Structure-activity relationship studies haverevealed that an increase in binding (T_(m)) of certain 2′-sugarmodified oligonucleotides to an RNA target (complement) correlates withan increased “A” type conformation of the heteroduplex. Furthermore,absolute fidelity of the modified oligonucleotides is maintained.Increased binding of 2′-sugar modified sequence-specificoligonucleotides of the invention provides superior potency andspecificity compared to phosphorus-modified oligonucleotides such asmethyl phosphonates, phosphate triesters and phosphoramidates as knownin the literature.

The only structural difference between DNA and RNA duplexes is ahydrogen atom at the 2′-position of the sugar moiety of a DNA moleculeversus a hydroxyl group at the 2′-position of the sugar moiety of an RNAmolecule (assuming that the presence or absence of a methyl group in theuracil ring system has no effect). However, gross conformationaldifferences exist between DNA and RNA duplexes.

It is known from X-ray diffraction analysis of nucleic acid fibers(Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504) andanalysis of crystals of double-stranded nucleic acids that DNA takes a“B” form structure and RNA takes the more rigid “A” form structure. Thedifference between the sugar puckering (C2′ endo for “B” form DNA andC3′ endo for “A” form RNA) of the nucleosides of DNA and RNA is themajor conformational difference between double-stranded nucleic acids.

The primary contributor to the conformation of the pentofuranosyl moietyis the nature of the substituent at the 2′-position. Thus, thepopulation of the C3′-endo form increases with respect to the C2′-endoform as the electronegativity of the 2′-substituent increases. Forexample, among 2′-deoxy-2′-haloadenosines, the 2′-fluoro derivativeexhibits the largest population (65%) of the C3′-endo form, and the2′-iodo exhibits the lowest population (7%). Those of adenosine (2′-OH)and deoxy-adenosine (2′-H) are 36% and 19%, respectively. Furthermore,the effect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoroadenosine) is furthercorrelated to the stabilization of the stacked conformation. Researchindicates that dinucleoside phosphates have a stacked conformation witha geometry similar to that of A—A but with a greater extent of base-baseoverlapping than A—A. It is assumed that the highly polar nature of theC2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an “A” structure.

Data from UV hypochromicity, circular dichromism, and ¹H NMR alsoindicate that the degree of stacking decreases as the electronegativityof the halo substituent decreases. Furthermore, steric bulk at the2′-position of the sugar moiety is better accommodated in an “A” formduplex than a “B” form duplex.

Thus, a 2′-substituent on the 3′-nucleotidyl unit of a dinucleosidemonophosphate is thought to exert a number of effects on the stackingconformation: steric repulsion, furanose puckering preference,electrostatic repulsion, hydrophobic attraction, and hydrogen bondingcapabilities. These substituent effects are thought to be determined bythe molecular size, electronegativity, and hydrophobicity of thesubstituent.

Studies with a 2′-OMe modification of 2′-deoxy guanosine, cytidine, anduridine dinucleoside phosphates exhibit enhanced stacking effects withrespect to the corresponding unmethylated species (2′-OH). In this case,it is believed that the hydrophobic attractive forces of the methylgroup tend to overcome the destablilizing effects of its steric bulk.

Melting temperatures (complementary binding) are increased with the2′-substituted adenosine diphosphates. It is not clear whether the3′-endo preference of the conformation or the presence of thesubstituent is responsible for the increased binding. However, greateroverlap of adjacent bases (stacking) can be achieved with the 3′-endoconformation.

While we do not wish to be bound by theory, it is believed that theaminooxyalkyl substituents of the present invention also result in thesugar pucker of the nucleoside being C3′-endo puckering.

Compounds of the invention can be utilized as diagnostics, therapeuticsand as research reagents and kits. They can be utilized inpharmaceutical compositions by adding an effective amount of anoligonucleotide of the invention to a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism can be contacted with an oligonucleotide of theinvention having a sequence that is capable of specifically hybridizingwith a strand of target nucleic acid that codes for the undesirableprotein.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligomer in accordance with the invention, commonly in apharmaceutically acceptable carrier, in doses ranging from 0.01 μg to100 g per kg of body weight depending on the age of the patient and theseverity of the disease state being treated. Further, the treatment maybe a single dose or may be a regimen that may last for a period of timewhich will vary depending upon the nature of the particular disease, itsseverity and the overall condition of the patient, and may extend fromonce daily to once every 20 years. Following treatment, the patient ismonitored for changes in his/her condition and for alleviation of thesymptoms of the disease state. The dosage of the oligomer may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, or if the disease statehas been ablated.

In some cases it may be more effective to treat a patient with anoligomer of the invention in conjunction with other traditionaltherapeutic modalities. For example, a patient being treated for AIDSmay be administered an oligomer in conjunction with AZT, or a patientwith atherosclerosis may be treated with an oligomer of the inventionfollowing angioplasty to prevent reocclusion of the treated arteries.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual oligomers, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to several years.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the oligomer is administered in maintenance doses,ranging from 0.01 μg to 100 g per kg of body weight, once or more daily,to once every several years.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

The present invention can be practiced in a variety of organisms rangingfrom unicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular machinery is susceptible to such therapeuticand/or prophylactic treatment. Seemingly diverse organisms such asbacteria, yeast, protozoa, algae, plant and higher animal forms,including warm-blooded animals, can be treated in this manner. Further,since each of the cells of multicellular eukaryotes also includes bothDNA-RNA transcription and RNA-protein translation as an integral part oftheir cellular activity, such therapeutics and/or diagnostics can alsobe practiced on such cellular populations. Furthermore, many of theorganelles, e.g. mitochondria and chloroplasts, of eukaryotic cells alsoinclude transcription and translation mechanisms. As such, single cells,cellular populations or organelles also can be included within thedefinition of organisms that are capable of being treated with thetherapeutic or diagnostic oligonucleotides of the invention. As usedherein, therapeutics is meant to include both the eradication of adisease state, killing of an organism, e.g. bacterial, protozoan orother infection, or control of aberrant or undesirable cellular growthor expression.

The current method of choice for the preparation of naturally occurringoligonucleotides, as well as modified oligonucleotides such asphosphorothioate oligonucleotides, is via solid-phase synthesis whereinan oligonucleotide is prepared on a polymer support (a solid support)such as controlled pore glass (CPG); oxalyl-controlled pore glass (see,e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527); TENTAGELSupport, (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34,3373); or POROS, a polystyrene resin available from PerceptiveBiosystems. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. Suitable solid phase techniques, includingautomated synthesis techniques, are described in F. Eckstein (ed.),Oligonucleotides and Analogues, a Practical Approach, Oxford UniversityPress, New York (1991).

Solid-phase synthesis relies on sequential addition of nucleotides toone end of a growing oligonucleotide chain. Typically, a firstnucleoside (having protecting groups on any exocyclic aminefunctionalities present) is attached to an appropriate glass beadsupport and activated phosphite compounds (typically nucleotidephosphoramidites, also bearing appropriate protecting groups) are addedstepwise to elongate the growing oligonucleotide. Additional methods forsolid-phase synthesis may be found in Caruthers U.S. Pat. Nos.4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418;and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069.

Solid supports according to the invention include controlled pore glass(CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., NucleicAcids Research 1991, 19, 1527), TentaGel Support—anaminopolyethyleneglycol derivatized support (see, e.g., Wright, et al.,Tetrahedron Letters 1993, 34, 3373) or Poros—a copolymer ofpolystyrene/divinylbenzene.

2′-Substituted oligonucleotides were synthesized by standard solid phasenucleic acid synthesis using an automated synthesizer such as Model 380B(Perkin-Elmer/Applied Biosystems) or MilliGen/Biosearch 7500 or 8800.Triester, phosphoramidite, or hydrogen phosphonate coupling chemistries(Oligonucleotides: Antisense Inhibitors of Gene Expression. M.Caruthers, p. 7, J. S. Cohen (Ed.), CRC Press, Boca Raton, Fla., 1989)are used with these synthesizers to provide the desiredoligonucleotides. The Beaucage reagent (J. Amer. Chem. Soc., 1990, 112,1253) or elemental sulfur (Beaucage et al., Tet. Lett., 1981, 22, 1859)is used with phosphoramidite or hydrogen phosphonate chemistries toprovide 2′-substituted phosphorothioate oligonucleotides.

The requisite 2′-substituted nucleosides (A, G, C, T (U), and othernucleosides having modified nucleobases and or additional sugarmodifications) are prepared, utilizing procedures as described below.

During the synthesis of nucleosides and oligonucleotides of theinvention, chemical protecting groups can be used to facilitateconversion of one or more functional groups while other functionalgroups are rendered inactive. A number of chemical functional groups canbe introduced into compounds of the invention in a blocked form andsubsequently deblocked to form a final, desired compound. In general, ablocking group renders a chemical functionality of a molecule inert tospecific reaction conditions and can later be removed from suchfunctionality in a molecule without substantially damaging the remainderof the molecule (Green and Wuts, Protective Groups in Organic Synthesis,2d edition, John Wiley & Sons, New York, 1991). For example, aminogroups can be blocked as phthalimido groups, as9-fluorenylmethoxycarbonyl (FMOC) groups, and withtriphenylmethylsulfenyl, t-BOC, benzoyl or benzyl groups. Carboxylgroups can be protected as acetyl groups. Representative hydroxylprotecting groups are described by Beaucage et al., Tetrahedron 1992,48, 2223. Preferred hydroxyl protecting groups are acid-labile, such asthe trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX)groups. Chemical functional groups can also be “blocked” by includingthem in a precursor form. Thus, an azido group can be used considered asa “blocked” form of an amine since the azido group is easily convertedto the amine. Representative protecting groups utilized inoligonucleotide synthesis are discussed in Agrawal, et al., Protocolsfor Oligonucleotide Conjugates, Eds, Humana Press; New Jersey, 1994;Vol. 26 pp. 1-72.

Among other uses, the oligonucleotides of the invention are useful in aras-luciferase fusion system using ras-luciferase transactivation. Asdescribed in International Publication Number WO 92/22651, publishedDec. 23, 1992 and U.S. Pat. Nos. 5,582,972 and 5,582,986, commonlyassigned with this application, the entire contents of each of the aboveU.S. patents are herein incorporated by reference, the ras oncogenes aremembers of a gene family that encode related proteins that are localizedto the inner face of the plasma membrane. Ras proteins have been shownto be highly conserved at the amino acid level, to bind GTP with highaffinity and specificity, and to possess GTPase activity. Although thecellular function of ras gene products is unknown, their biochemicalproperties, along with their significant sequence homology with a classof signal-transducing proteins known as GTP binding proteins, or Gproteins, suggest that ras gene products play a fundamental role inbasic cellular regulatory functions relating to the transduction ofextracellular signals across plasma membranes.

Three ras genes, designated H-ras, K-ras, and N-ras, have beenidentified in the mammalian genome. Mammalian ras genes acquiretransformation-inducing properties by single point mutations withintheir coding sequences. Mutations in naturally occurring ras oncogeneshave been localized to codons 12, 13, and 61. The most commonly detectedactivating ras mutation found in human tumors is in codon-12 of theH-ras gene in which a base change from GGC to GTC results in aglycine-to-valine substitution in the GTPase regulatory domain of theras protein product. This single amino acid change is thought to abolishnormal control of ras protein function, thereby converting a normallyregulated cell protein to one that is continuously active. It isbelieved that such deregulation of normal ras protein function isresponsible for the transformation from normal to malignant growth.

In addition to modulation of the ras gene, the oligonucleotides of thepresent invention that are specifically hybridizable with other nucleicacids can be used to modulate the expression of such other nucleicacids. Examples include the raf gene, a naturally present cellular genewhich occasionally converts to an activated form that has beenimplicated in abnormal cell proliferation and tumor formation. Otherexamples include those relating to protein kinase C (PKC) that have beenfound to modulate the expression of PKC, those related to cell adhesionmolecules such as ICAM, those related to multi-drug resistanceassociated protein, and viral genomic nucleic acids include HIV,herpesviruses, Epstein-Barr virus, cytomegalovirus, papillomavirus,hepatitis C virus and influenza virus (see U.S. Pat. Nos. 5,166,195,5,242,906, 5,248,670, 5,442,049, 5,457,189, 5,510,476, 5,510,239,5,514,577, 5,514,786, 5,514,788, 5,523,389, 5,530,389, 5,563,255,5,576,302, 5,576,902, 5,576,208, 5,580,767, 5,582,972, 5,582,986,5,591,720, 5,591,600 and 5,591,623, commonly assigned with thisapplication, the disclosures of which are herein incorporated byreference).

As will be recognized, the steps of the methods of the present inventionneed not be performed any particular number of times or in anyparticular sequence. Additional objects, advantages, and novel featuresof this invention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are intended to beillustrative and not intended to be limiting.

EXAMPLE 1Methyl-2-O-(2-ethylacetyl)-3,5-bis-O-(2,4-dichlorobenzyl)-α-D-ribofuranoside(3, FIG. 1)

Compound 2 (FIG. 1) (multigram quantities of 2 were prepared from 1 viathe literature procedure, Martin, P. Helv. Chem. Acta, 1995, 78,486-504) was dissolved in DMF (86 mL) with cooling to 5° C., and NaH(60% dispersion, 1.38 g, 34.38 mmol) was added. The reaction mixture wasstirred at 5° C. for 5 minutes then warmed to ambient temperature andstirred for 20 minutes after which time the reaction mixture was cooledto 5° C. and ethylbromoacetate (3.81 mL, 34.4 mmol) was added dropwiseresulting in the evolution of gas. The reaction mixture was allowed towarm to ambient temperature and stirred for 3 hours after which time themixture was cooled to 5° C. and the pH was adjusted to 3 with saturatedaqueous NH₄Cl. The solvent was evaporated in vacuo to give a syrup whichwas dissolved in EtOAc (200 mL), washed with water and then brine. Theorganic layer was separated, dried with MgSO₄, and the solvent wasevaporated in vacuo to give an oil. The oil was purified by flashchromatography using hexanes-EtOAc, 60:40, to give the title compound(3) as an oil (15.52 g, 95%). ¹H NMR (CDCl₃): δ7.58-7.18 (m, 6H), 5.05(d, J=3.8 Hz, 1H), 4.79 (q, J_(AB)=13.7 Hz, 2H), 4.57 (d, J=2.8 Hz, 2H),4.31-4.16 (m, 5H), 4.03 (m, 2H), 3.62 (d, 2H), 3.50 (s, 3H), 1.28 (t,3H). ¹³C NMR (CDCl₃): δ_(—)170.0, 134.2, 133.6, 133.5, 130.3, 129.8,129.1, 128.8, 127.1, 102.1, 81.4, 78.9, 76.6, 70.6, 70.0, 69.3, 67.6,61.0, 55.6, 14.2. Anal. Calcd for C₂₄H₂₆Cl₄O₇.H₂O: C, 49.17; H, 4.81.Found: C, 49.33; H, 4.31.

EXAMPLE 21-[2′-O-(2-ethylacetyl)-3′,5′-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranosyl]thymine(4, FIG. 1)

Thymine (6.90 g, 54.6 mmol) was suspended in anhydrous dichloroethane(136 mL) and bis-trimethylsilylacetamide (40.5 mL, 164 mmol) was added.The reaction mixture was heated to reflux temperature for 10 minutes togive dissolution. After cooling to ambient temperature, the solution wasadded to compound 3 with stirring. Trimethylsilyltrifluoromethanesulfonate (6.86 mL, 35.5 mmol) was added and thereaction mixture was heated to reflux for 6 hours. The mixture wascooled to 5° C. and the pH was adjusted to 7 by the slow addition ofsaturated NaHCO₃. The mixture was extracted with CH₂Cl₂ (3×150 mL) andthe organic extracts were combined, washed with brine, and the solventwas evaporated in vacuo to give an oil. The oil was dissolved in CH₂Cl₂and purified by flash chromatography using hexanes-EtOAc, 45:55, toprovide the title compound (4) as an oil (7.92 g, 44%). (The α-anomerwas contained in a later fraction). ¹H NMR (400 MHZ, CDCl₃): δ8.25 (s,1H), 7.67 (s, 1H), 7.46-7.21 (m, 6H), 5.94 (d, J=1.6 Hz, 1H), 4.80 (q,J_(AB=)12.4 Hz, 2H), 4.70-4.18 (m, 9H), 4.02 (d, 1H), 3.75 (d, 1H), 1.58(s, 3H), 1.26 (t, 3H). ¹³C NMR (CDCl₃): 5 170.1, 164.3, 150.3, 135.5,134.5, 134.2, 134.1, 133.8, 133.5, 130.7, 130.2, 129.4, 129.0, 127.1,110.3, 88.4, 80.8, 80.5, 74.7, 70.1, 68.9, 68.0, 66.2, 60.9, 14.1, 12.1.Anal. Calcd for C₂₈H₂₈Cl₄N₂O₈.H₂O: C, 49.43; H, 4.44; N, 4.12. Found: C,49.25; H, 4.10; N, 3.94.

EXAMPLE 31-[2′-O-(2-hydroxyethyl)-3′,5′-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranosyl]thymine(5, FIG. 1)

Compound 4 (9.92 g, 15.0 mmol) was dissolved in hot EtOH (150 mL) andthe solution was cooled to ambient temperature in a water bath. To thesolution was cautiously added NaBH₄ (1.13 g, 30.0 mmol) over 10 minutes.After 3 hours additional NaBH₄ (282 mg, 7.45 mmol) was added the mixturewas stirred for 1 hour and left to stand for 8 hours. The pH wasadjusted to 4 by addition of Saturated NH₄Cl (25 mL) to give a gum. Thesolvent was decanted and evaporated in vacuo to afford a white solidwhich was dissolved in CH₂Cl₂ (250 mL). The gum was dissolved withsaturated aqueous NaHCO₃ and this solution was gently extracted with theCH₂Cl₂ containing the product. The organic layer was separated and theaqueous layer was extracted again with CH₂Cl₂ (2×50 mL). After combiningthe organic layers, the solvent was dried over MgSO₄ and evaporated invacuo to afford a white foam. The foam was dissolved in CH₂Cl₂ andpurified by flash chromatography using hexanes-EtOAc, 20:80, to give thetitle compound (5) as a white foam (8.39 g, 90%). ¹H NMR (CDCl₃): δ10.18(s, 1H), 7.66 (s, 1H), 7.39-7.20 (m, 6H), 5.96 (s, 1H), 4.76-3.62 (m,14H), 1.58 (s, 3H). ¹³C NMR (CDCl₃): δ164.0, 150.8, 135.2, 134.6, 134.2,134.1, 133.5, 133.4, 130.2, 129.4, 129.0, 127.1, 110.6, 88.6, 81.0,80.7, 75.2, 72.0, 70.1, 68.9, 68.1, 61.9, 12.1.

EXAMPLE 41-[2′-O-(2-phthalimido-N-hydroxyethyl)-3′,5′-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranosyl]thymine(6, FIG. 1)

Compound 5 was dried by coevaporation with anhydrous acetonitrilefollowed by further drying in vacuo (0.1 torr) at ambient temperaturefor 12 h. The dried material (8.39 g, 13.53 mmol) was dissolved infreshly distilled THF (97 mL), PPh₃ (3.90 g, 14.9 mmol), andN-hydroxyphthalimide (2.43 g, 14.9 mmol) was added. The reaction mixturewas cooled to −78 ° C., and diethyl azodicarboxylate (2.34 mL, 14.9mmol) was added. The reaction mixture was warmed to ambient temperatureand the solvent was evaporated in vacuo to give a foam. The foam wasdissolved in EtOAc (100 mL) and washed with saturated aqueous NaHCO₃(3×30 mL). The organic layer was separated, washed with brine, driedover MgSO₄, and the solvent evaporated to give a foam. The foam waspurified by flash chromatography using CH₂Cl₂-acetone, 85:15, to givethe title compound (6) as a white foam (3.22 g, 31%). A secondchromatographic purification provided additional 6 as a white foam (5.18g, 50%). ¹H NMR (400 MHZ, CDCl₃): δ9.0 (s, 1H), 7.8 (m, 11H), 5.95 (s,1H), 4.84-3.70 (m, 13H), 1.60 (s, 3H). ¹³C NMR (100 MHZ, CDCl₃): δ163.7,163.5, 150.2, 138.0, 135.6, 134.5, 134.1, 134.0, 133.9, 133.7, 133.6,130.6. 130.4, 130.1, 129.8, 129.4, 129.1, 129.0, 128.8, 127.2, 123.5,110.4, 88.2, 81.0, 80.9, 77.6, 75.4, 70.2, 68.9, 68.4, 68.1., 12.1. LRMS(FAB+) m/z: 766 (M+H). LRMS (FAB−) m/z: 764 (M−H).

EXAMPLE 51-[2′-O-(2-phthalimido-N-oxyethyl)-3′,5′-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranosyl]thymine(7, FIG. 2)

Compound 6 (1.79 g, 2.34 mmol) was dissolved in CH₂Cl₂ (12 mL), thesolution was cooled to −78° C. and 1.0 M boron trichloride (5.15 mL,5.15 mmol) in CH₂Cl₂ was added and the reaction mixture was kept at 5°C. for 1.5 hours. Additional 1.0 M boron trichloride (5.15 mL, 5.15mmol) in CH₂Cl₂ was added and the solution was stirred at 5° for anadditional 1.5 hours. The pH was adjusted to 7 with saturated aqueousNaHCO₃ (30 mL). After dilution with CH₂Cl₂ (100 mL), the organic layerwas separated, and the aqueous layer was extracted with CHCl₃ (5×25 mL)and then EtOAc (3×25 mL). The organic layers were combined, dried overNa₂SO₄, and evaporated in vacuo to give an oil. The oil was purified byflash chromatography using CH₂Cl₂-acetone, 45:55, to provide the titlecompound (7) as a white foam (619 mg, 59%). ¹H NMR (CDCl₃): δ8.8 (br,1H), 7.88-7.75 (m, 4H), 7.50 (s, 1H), 5.70 (d, J=4 Hz, 1H), 4.45-3.75(m, 11H), 2.95 (br, 1H), 1.90 (s, 3H). ¹³C NMR (100 MHZ, CDCl₃): δ164.3,163.7, 150.6, 137.4, 134.7, 128.5, 123.6, 110.5, 89.7, 84.7, 81.9, 77.6,68.5, 68.4, 61.0, 12.3. LRMS (FAB+) m/z: 448 (M+H). LRMS (FAB−) m/z: 446(M−H).

EXAMPLE 61-[2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,41-dimethoxytrityl)-β-D-ribofuranosyl]thymine(8, FIG. 2)

Compound 7 was dried by coevaporation with anhydrous acetonitrilefollowed by further drying in vacuo (0.1 torr) at ambient temperaturefor 12 hours. The dried material (619 mg, 1.38 mmol) was dissolved inanhydrous pyridine (7 mL) and 4,4′-dimethoxytrityl chloride (514 mg,1.52 mmol) was added. After 2 hours additional 4,4′-dimethoxytritylchloride (257 mg, 0.76 mmol) was added. The solution was stirred for 2hours and a final addition of 4,4′-dimethoxytrityl chloride (257 mg,0.76 mmol) was made. After 12 h MeOH (10 mL) was added to the reactionmixture, it was stirred for 10 min and the solvent was evaporated invacuo to give an oil which was coevaporated with toluene. The oil waspurified by flash chromatography by pre-treating the silica withCH₂Cl₂-acetone-pyridine, 80:20:1, then using CH₂Cl₂-acetone, 80:20 toafford the title compound (8) as a yellow solid (704 mg, 68%). ¹H NMR(CDCl₃): δ7.8-6.8 (m, 18H), 5.94 (d, J=2.2 Hz, 1H), 4.57-4.12 (m, 7H),3.78 (s, 6H), 3.53 (m, 2H), 1.34 (s, 3H). ¹³C NMR (CDCl₃): δ164.3,163.8, 158.6, 150.6, 144.4, 135.5, 135.4, 134.7, 130.1, 128.7, 128.2,128.0, 127.1, 123.7, 113.3, 110.9, 87.9, 86.7, 83.2, 68.7, 68.5, 61.7,55.2, 11.9. LRMS (FAB+) m/z: 750 (M+H). LRMS (FAB−) m/z: 748 (M−H).Anal. Calcd for C₄₁H₃₉N₃O₁₁.H₂O: C, 65.14; H, 5.38; N, 5.47. Found: C,63.85; H, 5.16; N, 5.14. Anal. Calcd for C₄₁H₃₉N₃O₁₁: C, 65.68; H, 5.24;N, 5.60. Found: C, 65.23; H, 5.27; N, 5.45.

EXAMPLE 71-[2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]thymine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](9, FIG. 2)

Compound 8 was dried by coevaporation with anhydrous pyridine (2×20 mL),then further dried in vacuo (0.1 torr) at ambient temperature for 12hours. The dried material (704 mg, 0.939 mmol) was dissolved in CH₂Cl₂(9 mL), diisopropylamine tetrazolide (80.4 mg, 0.47 mmol) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.33 mL, 1.03mmol) with stirring. After 2 hours at ambient temperature additional2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.33 mL, 1.03mmol) was added and the solution was stirred for 20 hours. The solventwas evaporated in vacuo to give an oil which was purified by flashchromatography by pre-treating the silica with CH₂Cl₂-acetone-pyridine,85:15:1, then using CH₂Cl₂-acetone, 85:15 to afford the title compound(9) as an oil (704 mg, 68%). The product was coevaporated with anhydrousacetonitrile (2×30 mL) and CH₂Cl₂ (2×30 mL) to afford a yellow foam. ¹HNMR (CDCl₃): δ8.6 (br, 1H), 7.78-6.82 (m, 18H) , 6.06 (m, 1H), 4.6-3.3(m, 14H), 3.75 (s, 6H), 2.66 (m, 1H), 2.37 (m, 1H), 1.36 (s, 3H), 1.16(m, 12H). ³¹P NMR (CDCl₃): δ150.5, 151.2. LRMS (FAB+) m/z: 950 (M+H).LRMS (FAB−) m/z: 948 (M−H). Anal. Calcd for C₅₀H₅₆N₅O₁₂P.H₂O: C, 62.04;H, 6.04; N, 7.24. Found: C, 62.20; H, 5.94; N, 7.34.

EXAMPLE 82′-O-(2-ethylacetyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(11, FIG. 3)

Adenosine (30.00 g, 112 mmol) was dissolved in hot anhydrous DMF (600mL) and the solution was cooled to ambient temperature. NaH (60%dispersion oil, 4.94 g, 124 mmol) was added and the mixture was stirredwith a mechanical stirrer for 1 hour. The resulting suspension wascooled to 5° C. and ethylbromoacetate (13.7 mL, 124 mmol) was added. Theresulting solution was stirred for 12 hours at ambient temperature andthe solvent was evaporated in vacuo to give a residue which contained2′-O-(2-ethylacetyl)adenosine (10) and the putative 3′-O-isomer. Thismaterial was coevaporated with pyridine to give a foam which wasdissolved in anhydrous pyridine (400 mL).1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (39.52 mL, 124 mmol) wasadded and the solution was stirred for 24 hours at ambient temperature.The solvent was evaporated in vacuo to give an oil which was dissolvedin EtOAc (500 mL) and washed with brine three times. The organic layerwas separated, dried over MgSO₄, and the solvent was evaporated in vacuoto afford an oil. The oil was purified by flash chromatography usinghexanes-EtOAc, 80:20, to give the title compound (11) as an oil (14.63g, 22%). ¹H NMR (CDCl₃): δ8.26 (s, 1H), 8.07 (s, 1H), 6.20 (br s, 2H),4.91 (dd, J_(1′,2′)=4.7 Hz, J_(2′,3′)=9.3 Hz, 1H), 4.64-3.97 (m, 8H),1.22 (t, 3H), 1.05 (m, 28 H). ¹³C NMR (CDCl₃): δ170.0, 155.5, 152.8,149.0 139.3, 120.2, 88.6, 82.2, 81.1, 69.9, 68.3, 60.8, 60.0, 17.2,14.0, 12.7. Anal. Calcd for C₂₆H₄₅N₅O₇Si₂: C, 52.41; H, 7.61; N, 11.75,Si, 9.43. Found: C, 52.23; H, 7.34; N, 11.69.

EXAMPLE 92′-O-(2-hydroxyethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(12, FIG. 3)

Compound 11 (4.175 g, 7.01 mmol) was dissolved in ethanol (95%, 40 mL)and the resulting solution was cooled to 5° C. NaBH₄ (60% oildispersion, 0.64 g, 16.8 mmol) was added, and the mixture was allowed towarm to ambient temperature. After stirring for 12 hours CH₂Cl₂ (200 mL)was added and the solution was washed with brine twice and the organiclayer was separated. The organic layer was dried over MgSO₄, and thesolvent was evaporated in vacuo to give an oil. The oil was purified byflash chromatography using EtOAc-MeOH, 95:5, to afford the titlecompound (12) as an oil (0.368 g, 9.5%). ¹H NMR (CDCl₃): δ8.31 (s, 1H),8.14 (s, 1H), 6.18 (br s, 2H), 6.07 (s, 1H), 4.62 (dd, J_(1′,2′)=4.6 Hz,J_(2′,3′)=9.4 Hz, 1H), 4.3-3.5 (m, 8H), 1.03 (m, 28H). ¹³C NMR (CDCl₃):δ155.5, 153.0, 148.7, 138.3, 120.3, 89.2, 82.7, 81.4, 73.5, 69.3, 61.8,59.7, 17.2, 17.0, 16.8, 13.4, 12.9, 12.8, 12.6. LRMS (FAB+) m/z: 554(M+H), 686 (M+Cs+).

EXAMPLE 102′-O-(2-Phthalimido-N-hydroxyethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(13, FIG. 4)

To a solution of compound 12 (0.330 g, 0.596 mmol) in anhydrous THF (10mL) was added triphenylphosphine (0.180 g, 0.685 mmol) andN-hydroxyphthalimide (0.112 g, 0.685 mmol). To this mixture diethylazodicarboxylate (0.11 mL, 685 mmol) was added dropwise at 5° C. Afterstirring for 3 hours at ambient temperature, the solvent was evaporatedto give an oil. The oil was dissolved in EtOAc and washed with saturatedaqueous NaHCO₃ (×3) and brine. The organic layer was separated, driedover MgSO₄. The solvent was evaporated in vacuo to give an oil. The oilwas purified by flash chromatography using EtOAc-MeOH, 95:5, to give thetitle compound (13) as an oil (0.285 g, 68%). ¹H NMR (CDCl₃): δ8.21 (s,1H), 8.05 (s, 1H), 7.8-7.45 (m, 4H), 6.00 (s, 1H), 5.88 (br s, 2H), 4.92(dd, J_(1′,2′)=4.6, J_(2′,3′)=9.0 Hz), 4.5-3.9 (m, 8H), 1.0 (m, 28H).¹³C NMR (CDCl₃): δ163, 155.3, 152.8, 149, 139.6, 134.3, 123.4, 120,88.7, 82.7, 81.1, 77.4, 70.2, 69.5, 60.1, 17.4, 17.2, 17.0, 16.9, 13.3,12.9, 12.7, 12.6. LRMS (FAB+) m/z: 699 (M+H).

EXAMPLE 11N6-Benzoyl-2′-O-(2-phthalimido-N-hydroxyethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(14, FIG. 4)

To a solution of compound 13 (1.09 g, 1.97 mmol) in anhydrous pyridine(19 mL) cooled to 5° C. was added benzoyl chloride (1.14 mL, 9.8 mmol)and the resulting mixture was stirred at ambient temperature for 12hours. After cooling the mixture to 5° C., cold water (3.8 mL) wasadded, the mixture was stirred for 15 minutes, and conc NH₄OH (3.8 mL)was added. After stirring for 30 minutes at 5° C. the solvent wasevaporated to give a residue which was dissolved in water and extractedwith CH₂Cl₂ three times. The organic extracts were combined, dried overMgSO₄, and evaporated in vacuo to afford an oil. The oil was purified byflash chromatography using hexanes-EtOAc, 50:50, then 20:80, to give thetitle compound (14) as an oil (0.618 g, 48%). ¹H NMR (CDCl₃): δ9.2 (brs, 1H), 8.69 (s, 1H), 8.27 (s, 1H), 8.0-7.4 (m, 9H), 6.12 (s, 1H), 4.95(dd, J_(1′,2′)=4.7 Hz, J_(2′,3′)9.1 Hz, 1H), 4.5-4.0 (m, 8H), 1.06 (m,28H). ³C NMR (CDCl₃): δ164.4, 163.3, 152.5, 150.8, 149.3, 142.1, 134.4,133.7, 132.6, 132.1, 128.7, 128.2, 127.7, 123.4, 88.9, 82.7, 81.3, 77.5,70.1, 69.6, 60.0, 17.2, 17.0, 16.8, 13.3, 12.8, 12.7, 12.6. LRMS (FAB+)m/z: 803 (M+H).

EXAMPLE 12 N⁶-Benzoyl-2′-O-(2-phthalimido-N-hydroxyethyl)adenosine (15,FIG. 4)

To a solution of compound 14 (0.680 g, 0.847 mmol) in THF (20 mL) in apolyethylene reaction vessel at 5° C. was added HF-pyridine (70%, 0.48mL, 16.9 mmol) and the resulting mixture was warmed to ambienttemperature. After stirring for 12 hours the solvent was evaporated invacuo, EtOAc was added, the solution was washed with water, and theaqueous layer was separated and extracted with EtOAc. The organic layerswere combined, dried over MgSO₄, and the solvent was evaporated in vacuoto give the title compound (15) as a solid (408 mg, 86%). ¹H NMR(DMSO-d₆): δ11.2 (br s, 1H), 8.71 (s, 1H), 8.67 (s, 1H), 8.0-7.5 (m,9H), 6.11 (d, J 1′,2′=5.7 Hz), 5.23 (d, 1H), 5.14 (t, 1H), 4.66 (t, 1H),4.35 (m, 3H), 3.90 (m, 3H), 3.6 (m, 2H). ¹³C NMR (DMSO-d₆): δ163.5,152.0, 143.2, 135.0, 132.6, 131.9, 131.7, 129.3, 128.7, 128.5, 123.4,86.3, 85.8, 81.3, 76.8, 69.0, 68.7, 61.3. LRMS (FAB+) m/z: 561 (M+H, 583(M+Na+).

EXAMPLE 13N⁶-Benzoyl-2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,4′-dimethoxytrityl)adenosine(16, FIG. 4)

To a solution of compound 15 (0.258 g, 0.46 mmol) in anhydrous pyridine(5 mL) was added 4,4′-dimethoxytrityl chloride (0.179 g, 0.53 mmol) andthe solution was stirred for 12 hours at ambient temperature. Water wasadded and the mixture was extracted with EtOAc three times. The organicextracts were combined, evaporated in vacuo, and dried over MgSO₄. Theresulting oil was purified by flash chromatography using hexanes-EtOAc,90:10, to give the title compound (16) as an oil (0.249 g, 63%). ¹H NMR(CDCl₃): δ9.16 (br s, 1H), 8.68 (s, 1H), 8.28 (s, 1H), 8.1-6.8 (m, 22H),6.26 (d, J 1′,2′=4.0 Hz, 1H), 4.76 (m, 1H), 4.60 (m, 1H), 4.4-4.3 (m,3H), 4.13-4.0 (m, 3H), 3.77 (s, 6H), 3.48 (m, 2H). ¹³C NMR (CDCl₃):δ164.5, 163.6, 158.5, 152.6, 151.4, 149.5, 144.5, 141.9, 135.7, 134.7,132.7. 130.1, 128.8, 128.2, 127.8, 126.9, 123.7, 113.2, 87.2, 84.1,82.6, 69.9, 69.0, 63.0, 60.3, 55.2. HRMS (FAB+) m/z (M+Cs+) calcd forC₄₈H₄₂N₆O₁₀ 995.2017, found 995.2053 (M+Cs+).

EXAMPLE 14N⁶-Benzoyl-2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,4′-dimethoxytrityl)adenosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](17, FIG. 4)

To a solution of compound 16 (0.300 g, 0.348 mmol) in CH₂Cl₂ (10 mL) wasadded diisopropylamine tetrazolide (0.030 g, 0.174 mmol) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.13 mL, 0.418mmol). After stirring for 12 hours at ambient temperature additionaldiisopropylamine tetrazolide (0.060 g, 0.348 mmol) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.26 mL, 0.832mmol) were added in two portions over 24 hours. After 24 hoursCH₂Cl₂-NEt₃, 100:1, was added and the mixture was washed with saturatedaqueous NaHCO₃ and brine. The organic layer was separated, dried overMgSO₄, and the solvent was evaporated in vacuo. The resulting oil waspurified by flash chromatography by pre-treating the silica withhexanes-EtOAc-NEt₃, (40:60:1), then using the same solvent system togive the title compound (17) as an oil (203 g, 55%). ¹H NMR (CDCl₃):δ6.27 (m, 1H). ³¹P NMR (CDCl₃): δ151.0, 150.5. HRMS (FAB+) m/z (M+Cs+)calcd for C₅₇H₅₉N₈O₁₁P 1195.3095, found 1195.3046 (M+Cs+).

EXAMPLE 152′-O-(2-aminooxyethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(18, FIG. 5)

To a solution of compound 13 (0.228 g, 0.326 mmol) in CH₂Cl₂ (5 mL) at5° C. was added methylhydrazine (0.017 mL, 0.326 mmol) with stirring for2 hours. The mixture was filtered to remove a precipitate and thefiltrate was washed with water and brine. The organic layer wasseparated, dried over MgSO₄, and the evaporated in vacuo to give thetitle compound (18) as an oil (186 mg). The oil was of sufficient purityfor subsequent reactions. ¹H NMR (CDCl₃): δ8.31 (s, 1H), 8.15 (s, 1H),6.07 (s, 1H), 5.78 (br s, 2H), 4.70 (dd, J 1′,2′=4.4 Hz, J 2′,3′=9.0 Hz,1H), 4.3-3.9 (m, 8H), 1.9 (br, 2H), 1.0 (m, 28H). LRMS (FAB+) m/z: 569(M+H), 702 (M+Cs⁺).

EXAMPLE 162′-O-(2-O-Formaldoximylethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(19, FIG. 5)

To a solution of compound 18 (0.186 g, 0.326 mmol) in EtOAc (2 mL) andMeOH (2 mL) was added formaldehyde (aqueous 37%, 0.028 mL, 0.342 mmol)with stirring at ambient temperature for 3 hours. The solvent wasevaporated in vacuo to give the title compound (19) as an oil (189 mg).The oil was of sufficient purity for subsequent reactions. ¹H NMR(CDCl₃): δ8.31 (s, 1H), 8.09 (s, 1H), 6.97 (d, J=8.3 Hz, 1H), 6.38 (d,J=8.3 Hz, 1H), 6.01 (s, 1H), 5.66 (br s, 2H), 4.77 (dd, J_(1′,2′)=4.7Hz, J_(2′,3′)=9.3 Hz), 4.3-4.0 (m, 8H), 1.0 (m, 28H). LRMS (FAB+) m/z:581 (M+H), 713 (M+Cs⁺).

EXAMPLE 17N⁶-Benzoyl-2′-O-(2-O-formaldoximylethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine(20, FIG. 5)

To a solution of compound 19 (0.189 g, 0.326 mmol) in pyridine (5 mL) at5° C. was added benzoyl chloride (0.19 mL, 1.63 mmol) and the resultingsolution was stirred at ambient temperature for 3 hours. The solutionwas cooled to 5° C. and concentrated NH₄OH (1.5 mL) was added withstirring for 1 hour. The solvent was evaporated in vacuo to give an oilwhich was dissolved in CH₂Cl₂. The solution was washed with water andthe organic layer was separated, dried with MgSO₄, and the solvent wasevaporated to give the title compound (20) (223 mg) as an oil which wasof sufficient purity for subsequent reactions. ¹H NMR (CDCl₃): δ9.30(br, 1H), 8.79 (s, 1H), 8.31 (s, 1H), 8.1-7.2 (m, 5H), 7.00 (d, 1H),6.39 (d, 1H), 6.09 (s, 1H), 4.77 (dd, 1H), 4.4-3.9 (m, 8H), 1.1 (m,28H).

EXAMPLE 18 N⁶-Benzoyl-2′-O-(2-O-formaldoxinylethyl)adenosine (21, FIG.5)

To a solution of compound 20 (223 mg, 0.326 mmol) in THF (10 mL) in apolyethylene reaction vessel at 5° C. was added HF-pyridine (70%, 0.19mL, 6.5 mmol) and the mixture was allowed to warm to ambienttemperature. After stirring for 48 hours the solvents were evaporated invacuo to give a residue which was dissolved in EtOAc and washed withwater. The organic layer was separated, the aqueous layer was extractedwith EtOAc, and the organic layers were combined, dried over MgSO₄, andevaporated in vacuo. The resulting residue was purified by flashchromatography using EtOAc-MeOH, 95:5, to give the title compound (21)as a solid (24 mg, 17% from 13). ¹H NMR (CDCl₃): δ9.05 (br s, 1H), 8.77(s, 1H), 8.13 (s, 1H), 7.9-7.2 (m), 6.26 (d, J=10.7 Hz, 1H), 6.03 (d,J_(1′,2′)=7.8 Hz), 4.88 (dd, J=4.6 Hz, J=7.9 Hz, 1H), 4.6-3.7 (m, 10H).LRMS (FAB+) m/z: 443 (M+H). LRMS (FAB−) m/z: 441 (M−H).

EXAMPLE 19N6-Benzoyl-2′-O-(2-O-formaldoximylethyl)-5′-O-(4,4′-dimethoxytrityl)adenosine(21A, FIG. 5)

To a solution of compound 21 (0.34 g, 0.768 mmol) in pyridine (7 mL) wasadded 4,4′-dimethoxytrityl chloride (0.312 g, 0.922 mmol) and thereaction mixture was stirred at ambient temperature for 5 hours.Additional amounts of 4,4′-dimethoxytrityl chloride (520 mg, 1.54 mmoland 340 mg, 0.768 mmol) were added over 24 hours. The solvent wasevaporated, the crude product was dissolved in EtOAc, and washed withwater. The organic layer was separated, dried over MgSO₄ and the solventwas evaporated in vacuo. The crude material was purified by columnchromatography using EtOAc-Hexanes-NEt₃, 80:20:0.5, v/v/v, followed by,EtOAc-NEt3, 100:0.5, v/v, as solvent to give the title compound (21A) asan oil (0.269 g, 47%). ¹H NMR (CDCl₃): δ8.99 (br s, 1H), 8.74 (s, 1H),8.1-6.8 (m, 18H), 7.00 (d, 1H), 6.43 (d, 1H), 6.19 (d, 1H), 4.72 (m,1H), 4.48 (m, 1H), 4.23 (m, 3H), 4.1 (m, 1H), 3.9 (m, 1H), 3.78 (s, 6H),3.45 (m, 2H), 3.15 (d, 1H). HRMS (FAB+) m/z (M+Cs+) calcd for C₄₁H₄₀N₆O₈877.1962, found 877.1988 (M+Cs+).

EXAMPLE 20 2′-O-Allyl-5′-O-dimethoxytrityl-5-methyluridine

In a 100 mL stainless steel pressure reactor, allyl alcohol (20 mL) wasslowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10mmol) with stirring. Hydrogen gas rapidly evolved. Once the rate ofbubbling subsided, 2,2′-anhydro-5-methyluridine (1.0 g, 0.4.2 mmol) andsodium bicarbonate (6 mg) were added and the reactor was sealed. Thereactor was placed in an oil bath and heated to 170° C. internaltemperature for 18 hours. The reactor was cooled to room temperature andopened. Tlc revealed that all the starting material was gone (startingmaterial and product Rf 0.25 and 0.60 respectively in 4:1 ethylacetate/methanol on silica gel). The crude solution was concentrated,coevaporated with methanol (50 mL), boiling water (15 mL), absoluteethanol (2×25 mL) and then the residue was dried to 1.4 g of tan foam (1mm Hg, 25° C., 2 hours). A portion of the crude nucleoside (1.2 g) wasused for the next reaction step without further purification. Theresidue was coevaporated with pyridine (30 mL) and redissolved inpyridine (30 mL). Dimethoxytrityl chloride (1.7 g, 5.0 mmol) was addedin one portion at room temperature. After 2 hours the reaction wasquenched with methanol (5 mL), concentrated in vacuo and partitionedbetween a solution of saturated sodium bicarbonate and ethyl acetate(150 mL each). The organic phase was separated, concentrated and theresidue was subjected to column chromatography (45 g silica gel) using asolvent gradient of hexanes-ethyl acetate-triethylamine (50:49:1) to(60:39:1). The product containing fractions were combined, concentrated,coevaporated with acetonitrile (30 mL) and dried (1 mm hg , 25° C., 24hours) to 840 mg (34% two-step yield) of white foam solid. The NMR wasconsistent with the unmethylated uridine analog reported in theliterature.

EXAMPLE 21 2′-O-(2-hydroxyethyl)-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Allyl-5′-O-dimethoxytrityl-5-methyluridine (1.0 g, 1.6 mmol),aqueous osmium tetroxide (0.15 M, 0.36 mL, 0.0056 mmol, 0.035 eq) arid4-methylmorpholine N-oxide (0.41 g, 3.5 mmol, 2.15 eq) were dissolved indioxane (20 mL) and stirred at 25° C. for 4 hours. Tlc indicatedcomplete and clean reaction to the diol (Rf of starting to diol 0.40 to0.15 in dichloromethane/methanol 97:3 on silica). Potassium periodate(0.81 g, 3.56 mmol, 2.2 eq) was dissolved in water (10 mL) and added tothe reaction. After 17 hours the tlc indicated a 90% complete reaction(aldehyde Rf 0.35 in system noted above). The reaction solution wasfiltered, quenched with 5% aqueous sodium bisulfite (200 mL) and theproduct aldehyde was extracted with ethyl acetate (2×200 mL). Theorganic layers were combined, washed with brine (2×100 mL) andconcentrated to an oil. The oil was dissolved in absolute ethanol (15mL) and sodium borohydride (1 g) was added. After 2 hours at 25° C. thetlc indicated a complete reaction. Water (5 mL) was added to destroy theborohydride. After 2 hours the reaction was stripped and the residue waspartitioned between ethyl acetate and saturated sodium bicarbonatesolution (50 mL each). The organic layer was concentrated in vacuo andthe residue was columned (silica gel 30 g, dichloromethane-methanol97:3). The product containing fractions were combined and stripped anddried to 0.50 g (50%) of white foam. The NMR was consistent with that ofmaterial prepared by the glycosylation route.

EXAMPLE 22 2′-O-(2-hydroxyethyl)-5-methyluridine

In a 100 mL stainless steel pressure reactor, ethylene glycol (20 mL)was slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL,10 mmol) with stirring. Hydrogen gas rapidly evolved. Once the rate ofbubbling subsided, 2,2′-anhydro-5-methyluridine (1.0 g, 0.4.2 mmol) andsodium bicarbonate (3 mg) were added and the reactor was sealed. Thereactor was placed in an oil bath and heated to 150° C. internaltemperature for 72 hours. The bomb was cooled to room temperature andopened. TLC revealed that 65% of the starting material was gone(starting material and product Rf 0.25 and 0.40 respectively in 4:1ethyl acetate/methanol on silica gel). The reaction was worked upincomplete. The crude solution was concentrated (1 mm Hg at 100° C.,coevaporated with methanol (50 mL), boiling water (15 mL) and absoluteethanol (2×25 mL) and the residue was dried to 1.3 g of off-white foam(1 mm Hg, 25° C., 2 hours). NMR of the crude product was consistent with65% desired product and 35% starting material. The TLC Rf matched (oncospot) the same product generated by treating the DMT derivative abovewith dilute hydrochloric acid in methanol as well as the Rf of one ofthe spots generated by treating a sample of this product withdimethoxytrityl chloride matched the known DMT derivative (other spotswere DMT on side chain and bis substituted product).

EXAMPLE 23N4-benzoyl-2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,4′-dimethoxytrityl)cytidine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](25, FIG. 6)

The 2′-O-aminooxyethyl cytidine and guanosine analogs may be preparedvia similar chemistry in combination with reported literatureprocedures. Key to the synthetic routes is the selective 2′-O-alkylationof unprotected nucleosides. (Guinosso, C. J., Hoke, G. D., Frier, S.,Martin, J. F., Ecker, D. J., Mirabelli, C. K., Crooke, S. T., Cook, P.D., Nucleosides Nucleotides, 1991, 10, 259; Manoharan, M., Guinosso, C.J., Cook, P. D., Tetrahedron Lett., 1991, 32, 7171; Izatt, R. M.,Hansen, L. D., Rytting, J. H., Christensen, J. J., J. Am. Chem. Soc.,1965, 87, 2760. Christensen, L. F., Broom, A. D., J. Org. Chem., 1972,37, 3398. Yano, J., Kan, L. S., Ts'o, P. O. P., Biochim. Biophys. Acta,1980, 629, 178; Takaku, H., Kamaike, K., Chemistry Lett. 1982, 189).Thus, cytidine may be selectively alkylated to afford the intermediate2′-O-(2-ethylacetyl)cytidine 22. The 3′-isomer of 22 is typicallypresent in a minor amount and can be resolved by chromatography orcrystallization. Compound 22 can be protected to give2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)cytidine (23). Reductionof the ester 23 should yield2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)cytidine (24) which canbe N-4-benzoylated, the primary hydroxyl group may be displaced byN-hydroxyphthalimide via a Mitsunobu reaction, and the protectednucleoside may be phosphitylated as usual to yieldN4-benzoyl-2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,4′-dimethoxytrityl)cytidine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](25).

EXAMPLE 24N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](31)

In a similar fashion the 2′-O-aminooxyethyl guanosine analog may beobtained by selective 2′-O-alkylation of diaminopurine riboside(multigram quantities of diaminopurine riboside may be purchased fromSchering AG (Berlin) to provide 2′-O-(2-ethylacetyl)diaminopurineriboside 26 along with a minor amount of the 3′-O-isomer. Compound 26may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine 27 bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., PCT Int. Appl., 85 pp.; PIXXD2; WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine 28 and2N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine29 which may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine(30). As before the hydroxyl group may be displaced byN-hydroxyphthalimide via a Mitsunobu reaction, and the protectednucleoside may phosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](31).

EXAMPLE 25 -(1-hydroxyphthalimido)-5-hexene (32, FIG. 9)

To a stirred solution of 5-hexane-1-ol (20 g, 0.2 mol) in THF (500 mL)was added triphenylphosphine (80 g, 0.3 mol) and N-hydroxyphthalimide(49 g, 0.3 mol). The mixture was cooled to 0° C. and diethylazidocarboxylate (48 mL, 0.3 mol) was added slowly over a period of 1 hour.The reaction mixture was allowed to warm to room temperature and theyellow solution was stirred overnight. The solvent was then evaporatedto give a yellow oil. The oil was dissolved in CH₂Cl₂ and washed withwater, saturated NaHCO₃ solution followed by a saturated NaCl solution.The organic layer was concentrated in vacuo and the resulting oil wasdissolved in a solution of CH₂Cl₂/ether to crystallize out Ph₃P═O asmuch as possible. After three steps of purification the title compoundwas isolated as a yellow waxy solid (yield 93%). ¹³C NMR: δ21.94, 24.83,27.58, 33.26,. 78.26, 114.91, 123.41, 128.40, 128.54, 128.63, 134.45 and163.8 ppm.

EXAMPLE 26 N-(1-hydroxyphthalimido-5,6-hexane-diol) (33, FIG. 9)

Compound 32 (2.59 g, 10 mmol), aqueous osmium tetroxide (0.15 M, 3.6 mL,0.056 mmol) and N-methylmorpholine-N-oxide (2.46 g, 21 mmol) weredissolved in THF (100 mL). The reaction mixture was covered withaluminum foil and stirred at 25° C. for 4 hours. Tlc indicated the diolwas formed. The solvent was evaporated and the residue was partitionedbetween water and CH₂Cl₂. The organic layer was washed with a saturatedsolution of NaCl and dried over anhydrous MgSO₄. Concentration of theorganic layer resulted in a brownish oil that was characterized by ¹³CNMR and used in the next step without further purification. ¹³C NMR:δ21.92, 28.08, 32.62, 66.76, 71.96, 78.33, 123.43, 128.47, 128.71,131.93, 132.13, 134.49, 163.89.

EXAMPLE 27 N-1-hydroxy phthalimido-6-O-dimethyoxytrityl-5,6 hexane-diol(34, FIG. 9)

The product from the previous step (3.0 g) was coevaporated withpyridine (2×20 mL) and dissolved in pyridine (100 mL). Dimethyoxytritylchloride (3.5 g, 10 mmol) was dissolved in of pyridine (30 mL) and addedto the diol dropwise over a period of 30 minutes. After 4 hours, thereaction was quenched with methanol (10 mL). The solvent was evaporatedand the residual product portioned between saturated sodium bicarbonatesolution and CH₂Cl₂ (100 mL each). The organic phase was dried overanhydrous MgSO₄, concentrated and the residue was subjected to silicagel flash column chromatography using hexanes-ethyl acetate-triethylamine (60:39:1). The product containing fractions were combined,concentrated in vacuo and dried to give a yellow foamy solid. NMRanalysis indicated the title compound as a pure homogenousdimethyoxytritylated solid (5.05 g, 83% yield).

EXAMPLE 28 (35, FIG. 9)

Compound 34 was phosphitylated (1.5 g, 2.5 mmol) in CH₂Cl₂ solvent (20mL) by the addition of Diisopropylamine tetrazolide (214 mg, 1.25 mmol)and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite (1.3 mL,4.0 mmol). After stirring the solution overnight the solvent wasevaporated and the residue was applied to silica column and eluted withhexanes-ethyl acetate-triethylamine (50:49:1). Concentration of theappropriate fractions gave 1.61 g of the phosphitylated compound as ayellow foam (81%).

EXAMPLE 29 Attachment of O—N linker to CPG (36, FIG. 10)

Succinylated and capped CPG was prepared according to method describedby P. D. Cook et al. (U.S. Pat. No. 5,541,307). Compound 34 (0.8 mmol),dimethylaminopyridine (0.2 mmol), 2.0 g of succinylated and capped CPGtriethylamine (160 μL) and DEC (4.0 mmol) were shaken together for 24hours. Pentachlorophenyl (1.0 mmol) was then added and the resultingmixture was shaken for 24 hours. The CPG beads were filtered off andwashed thoroughly with pyridine (30 mL) dichloromethane (2×30 mL), CH₃OH(30 mL) in ether. The CPG solid support was dried over P₂O₅ and itsloading was determined to be 28 μmols/g.

EXAMPLE 30 Synthesis of oligonucleotides using ON linker

The following oligonucleotides were synthesized using compound 35, whichis shown as an X at the 5′ end of the oligonucleotide:

SEQ ID NO:1 5′ XTTTTTTTTTT 3′

SEQ ID NO:2 5′ X TGC ATC CCC CAG GCC ACC ATT TTT T 3′

These oligonucleotides were synthesized as phosphorothioates. Compound35 was used as a 0.1 M solution in CH₃CN. The coupling efficiency ofON-linker was >95% as shown by trityl colors. The oligonucleotides wereretained in the solid support for solid phase conjugation.

EXAMPLE 31 Conjugation of pyrene to oligonucleotides using ON-linker

Oligonucleotide SEQ ID NO:1 in CPG (1 μmol) was taken in a glass funnelreactor and of 5% methylhydrazine (5 mL) in 9:1 CH₂Cl₂/CH₃OH was added.The reactor was shaken for 30 minutes. The methyl hydrazine was drained,washed with CH₂Cl₂ and the methyl hydrazine reaction was repeated. Thebeads were washed with CH₂Cl₂ followed by ether and dried. Pyrenebutyric acid-N-hydroxy succinimide (110 mg) in DMF (5 mL) was added.After shaking for 2 hours, the pyrene butyrate solution was drained, theoligonucleotide was deprotected in NH₄OH for 30 minutes at roomtemperature. The aqueous solution was then filtered and an HPLC analysiswas run. The product peak had a retention time of 34.85 minutes and thediode-array spectrophotometer showed pyrene absorption.

EXAMPLE 32 Conjugation of pyrene butyraldehyde to oligonucleotide (SEQID NO:2)

Pyrene butyraldehyde is added to SEQ ID NO:2 after MeNHNH₂ treatment.NaCNBH₃ in MeOH was then added. Deprotection of CPG followed by NH₄OHcleaving of CPG showed pyrene conjugation to oligonucleotide.

EXAMPLE 33

To a stirred solution of 1,6-hexane-diol N-hydroxyphthalimide (6.525 g,0.039 mol) and triphenylphosphine (10.2 g, 0.039 mol) in anhydrous THF(100 mL) was added diethylazidocarboxylate (DEAD, 7.83 g, 0.045 mol)over a period of 1 hour at 5° C. under an atmosphere of argon. Thereaction mixture was then stirred at room temperature overnight. Thebright yellow solution was concentrated under vacuum to remove the THFand portioned between CH₂Cl₂ and water. The organic layer was thenwashed with saturated NaHCO₃ followed by saturated NaCl. It was thendried over anhydrous MgSO₄ and applied to a silica column and elutedwith EtOAC/hexane 1:1 to give 9.8 g. The material was contaminated withPh₃P═O and was recrystallized with CH₂Cl₂/ether. cl EXAMPLE 34

(FIGS. 11 and 12)

5-hexene-1-ol is sylylated using imidazole/TBDPS-Cl in CH₂Cl₂ to givecompound 37. Compound 37 is then dihydroxylated with OSO₄/NMMO as in(Example 25 for Compound 33) to give compound 38. Compound 38 isdimethoxytritylated at the primary alcohol function to give compound 39.It is then subjected to Mitsunobu reaction with N-hydroxyphthalimide togive compound 40. Compound 40 is then disilylated with TBAF (tetrabutylammonium fluoride, 1M in THF) to give compound 41. Compound 41 is thenderivatized to a phosphoramidite 42. Compound 41 was also separatelyconnected to controlled pore glass beads (Compound 43).

EXAMPLE 35

2,6,9-(β-D-ribofuranosyl) purine (5.64 g, 20 mmol) was added to asuspension of 800 mg of 60% sodium hydride in oil previously washed withhexanes in 100 mL of DMF under argon. After 1 hour of stirring at roomtemperature allyl bromide (2 mL, 1.1 equivalent) was added to thesolution and stirred at room temperature overnight. The reaction mixturewas evaporated and applied to a silica column and eluted withCH₂Cl₂/CH₃OH (20:1) containing 1% triethylamine. The total yield of 2′and 3′ O-allyl compounds was 5.02 g (77%). The mixture of 2′ and 3′isomers was then exocyclic amine protected by treatment of DMF DMA inMeoH in quantitative yield. This material was then5′-O-dimethyoxytritylated to give a mixture of5′-O-dimethoxytrityl-N-2-formamidine-2′-O-(2-hydroxy ethyl)-guanosineand 5′-O-dimethoxytrityl-N2-formamidine-3′-O-(2-hydroxyethyl)guanosinein 2:1 ratio. The final compounds were purified by silica gel flashcolumn chromatography.

EXAMPLE 36

2,6-diamino-9-(b-D-ribofuranosyl)purine (282 mg, 1 mmol) was added to asuspension of 40 mg of 60% sodium hydride in oil previously washed withhexanes in anhydrous DMF (5 mL). To this solution of2-(bromoethoxy)-t-butyldimethyl silane (220 mL) was added. The mixturewas stirred at room temperature overnight. The reaction mixture wasevaporated and the resulting oil was partitioned between water and ethylacetate. The organic layer was dried over Na₂SO₄. The reaction mixturewas purified to give the 2′ and 3′ isomers over the silica gel. The2′-material was then amine protected with DMF DMA and5′-dimethoxytrilated to give5′-O-dimethoxytrityl-N2-formamidine-2′-O-(2-TBDMS-hydroxyethyl)guanine.

EXAMPLE 37 Oligonucleotide Synthesis

Unsubstituted and substituted oligonucleotides are synthesized on anautomated DNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine. For phosphorothioateoligonucleotides, the standard oxidation bottle is replaced by 0.2 Msolution of 3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile forthe step wise thiation of the phosphite linkages. The thiation wait stepis increased to 68 sec and is followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hours), the oligonucleotides are purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Analytical gel electrophoresis is accomplished in 20%acrylamide, 8 M urea, 454 mM Tris-borate buffer, pH=7.0.Oligonucleotides and phosphorothioates are judged, based onpolyacrylamide gel electrophoresis, to be greater than 80% full-lengthmaterial.

EXAMPLE 38 General procedure for the attachment of2′-deoxy-2′-substituted 5′-dimethoxytriphenylmethyl ribonucleosides tothe 5′-hydroxyl of nucleosides bound to CPG support

The 2′-deoxy-2′-substituted nucleoside that will reside at the terminal3′-position of the oligonucleotide is protected as a 5′-DMT group (thecytosine and adenine exocyclic amino groups are benzoylated and theguanine amino is isobutrylated) and treated with trifluoroaceticacid/bromoacetic acid mixed anhydride in pyridine anddimethylaminopyridine at 50° C. for five hours. The solution is thenevaporated under reduced pressure to a thin syrup which is dissolved inethyl acetate and passed through a column of silica gel. The homogenousfractions are collected and evaporated to dryness. A solution of 10 mLof acetonitrile, 10 μM of the 3′-O-bromomethylester-modified nucleoside,and 1 mL of pyridine/dimethylaminopyridine (1:1) is syringed slowly (60to 90 sec) through a 1 μM column of CPG thymidine (Applied Biosystems,Inc.) that had previously been treated with acid according to standardconditions to afford the free 5′-hydroxyl group. Other nucleoside-boundCPG columns may be employed. The eluent is collected and syringed againthrough the column. This process is repeated three times. The CPG columnis washed slowly with 10 mL of acetonitrile and then attached to an ABI380B nucleic acid synthesizer. Oligonucleotide synthesis is nowinitiated. The standard conditions of concentrated ammonium hydroxidedeprotection that cleaves the thymidine ester linkage from the CPGsupport also cleaves the 3′,5′ ester linkage connecting the pyrimidinemodified nucleoside to the thymidine that was initially bound to the CPGnucleoside. In this manner, any 2′-substituted nucleoside or generallyany nucleoside with modifications in the heterocycle and/or sugar can beattached at the 3′ end of an oligonucleotide.

EXAMPLE 39 Modified oligonucleotide synthesis for incorporation of2′-substituted nucleotides

A. ABI Synthesizer

Oligonucleotide sequences incorporating1-[2′-O-(2-phthalimido-N-oxyethyl)-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]thyminewere synthesized on an ABI 380B utilizing phosphoramidite chemistry withdouble-coupling and increased coupling times (5 and 10 min). The2′-O-aminooxyethoxy phosphoramidite was used at a starting concentrationof 0.08 M and 10% tert-butyl peroxide in acetonitrile was used as theoxidizer. Deoxy phosphoramidites were used at 0.2 M. Finalconcentrations of 2′-O-aminooxyethoxy and deoxy amidites were 0.04 M and0.1 M, respectively. The oligonucleotides were cleaved from the CPGsupport and the base protecting groups removed using concentratedammonia at 55° C. for 6 hours. The oligonucleotides were purified bysize exclusion chromatography over Sephadex G25 and analyzed byelectrospray mass spectrometry and capillary gel electrophoresis. Deoxyphosphoramidites were purchased from Perseptive Biosystems GmbH.

(SEQ ID NO:5) CTC GTA CCt TTC CGG TCC. LRMS (ES−) m/z: calcd: 5453.2;found: 5453.5.

(SEQ ID NO:6) CTC GTA Ctt ttC CGG TCC. LRMS (ES−) m/z: calcd: 5693.2;found: 5692.9.

(SEQ ID NO:3) GCG ttt ttt ttt tGC G. LRMS (ES−) m/z: calcd: 5625.7;found: 5625.9.

B. Expedite Synthesizer

Oligonucleotides incorporating1-[2′-O-(2-phthalimdo-N-oxyethyl-5′-O-dimethoxytrityl-B-D-riboframosyl)-6-N-benzoyl-thyminewere synthesized in an Expedite 8690 Synthesizer. 130 mg of the amiditewas dissolved in dry CH₃CN (1.3 mL, app. 0.08M). 10% t-BuOOH in CH₃CNv/v was used as the oxidizing agent. An extended coupling and waitingtimes were used and a 10 min. oxidation was employed. Theoligonucleotide synthesis revealed excellent coupling yields (>98%).Oligonucleotides were purified and their mass spec and profilesdetermined.

Oligonucleo- SEQ ID tide Sequence NO: V CTC GTA CCa TTC CGG TCC 7 VI GGaCCG Gaa GGT aCG aG 8 VII aCC GaG GaT CaT GTC GTa CGC 9

where a represents1-[2′-O-(2-aminooxyethy1)-β-D-ribofuranosyl]adenosine. !

!

EXAMPLE 40 Oligonucleotide Having 2′-Substituted OligonucleotidesRegions Flanking Central 2′-Deoxy Phosphorothioate OligonucleotideRegion

A 15 mer RNA target of the sequence 5′GCGTTTTTTTTTTGCG 3′ (SEQ ID NO:3)is prepared in the normal manner on the DNA sequencer using RNAprotocols. A series of complementary phosphorothioate oligonucleotideshaving 2′-O-substituted nucleotides in regions that flank a 2′-deoxyregion are prepared utilizing 2′-O-substituted nucleotide precursorsprepared as per known literature preparations, i.e. 2′-O-methyl, or asper the procedure of International Publication Number WO 92/03568,published Mar. 5, 1992. The 2′-O-substituted nucleotides are added astheir 5′-O-dimethoxytrityl-3′-phosphoramidites in the normal manner onthe DNA synthesizer. The complementary oligonucleotides have thesequence of 5′ CGCAAAAAAAAAAAAACGC 3′ (SEQ ID NO:4). The2′-O-substituent is located in CGC and CG regions of theseoligonucleotides. The 2′-O-substituents used are 2′-aminooxyethyl,2′-O-ethylaminooxyethyl and 2′-O-dimethylaminooxyethyl.

EXAMPLE 41 Hybridization Analysis

A. Evaluation of the thermodynamics of hybridization of 2′-modifiedoligonucleotides.

The ability of the 2′-modified oligonucleotides to hybridize to theircomplementary RNA or DNA sequences is determined by thermal meltinganalysis. The RNA complement is synthesized from T7 RNA polymerase and atemplate-promoter of DNA synthesized with an Applied Biosystems, Inc.380B RNA species is purified by ion exchange using FPLC (LKB Pharmacia,Inc.). Natural antisense oligonucleotides or those containing2′-modifications at specific locations are added to either the RNA orDNA complement at stoichiometric concentrations and the absorbance (260nm) hyperchromicity upon duplex to random coil transition is monitoredusing a Gilford Response II spectrophotometer. These measurements areperformed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, andNaCl to yield an ionic strength of 10 either 0.1 M or 1.0 M. Data isanalyzed by a graphic representation of 1/T_(m) vs ln(Ct), where (Ct)was the total oligonucleotide concentration. From this analysis thethermodynamic para-meters are determined. Based upon the informationgained concerning the stability of the duplex of heteroduplex formed,the placement of modified pyrimidine into oligonucleotides are assessedfor their effects on helix stability. Modifications that drasticallyalter the stability of the hybrid exhibit reductions in the free energy(delta G) and decisions concerning their usefulness as antisenseoligonucleotides are made.

As is shown in the following table (Table 1), the incorporation of2′-substituted nucleosides of the invention into oligonucleotides canresult in significant increases in the duplex stability of the modifiedoligonucleotide strand (the antisense strand) and its complementary RNAstrand (the sense strand). The stability of the duplex increased as thenumber of 2′-substituted nucleosides in the antisense strand increased.As is evident from Table 1 the addition of a 2′-substituted nucleoside,irrespective of the individual nucleoside or the position of thatnucleoside in the oligonucleotide sequence, resulted in an increase inthe duplex stability.

In Table 1, the small case nucleosides represent nucleosides thatinclude substituents of the invention. Effects of 2′-O-aminooxyethoxymodifications on DNA(antisense)—RNA(sense) duplex stability.

TABLE 1 SEQ ID NO: Sequence 5 CTC GTA CCT TTC CGG TCC 5 CTC GTA CCt TTCCGG TCC 6 CTC GTA CTT TTC CGG TCC 6 CTC GTA Ctt ttC CGG TCC 3 GCG TTTTTT TTT TGC G 3 GCG ttt ttt ttt tGC G 3 GCG TTT TTT TTT TGC G* 3 GCG tttttt ttt tGC G* 7 CTC GTA CCa TTC CGG TCC 8 GGa CCG Gaa GGT aCG aG 9 aCCGaG GaT CaT GTC GTa CGC t =1-[2′-O-(2-aminooxyethyl)-β-D-ribofuranosyl]thymine. a =1-[2′-O-(2-aminooxyethyl)-β-D-ribofuranosyl]adenosine. * = washybridized against DNA as sense strand.

SEQ ID NO: subs. T_(m) ° C. ΔT_(m) ° C. ΔT_(m) ° C./sub. 5 0 65.2 ± 0.05 1 64.8 ± 0.1 −0.5 ± 0.1 −0.5 ± 0.1 6 0 61.5 ± 0.0 6 4 65.6 ± 0.4   4.1± 0.4   1.0 ± 0.1 3 0 48.2 ± 0.6 3 10  60.0 ± 0.0 11.9 ± 0.7 1.19 ± 0.07 3* 0 53.5 ± 0.1†  3* 10  44.0 ± 0.2† −9.4 ± 0.3† −.94 ± 0.03† In Table1, “subs.” = Number of substitutions, as described above.

As is evident from Table 1, the duplexes formed between RNA andoligonucleotides containing 2′-substituents of the invention exhibitedincreased binding stability as measured by the hybridizationthermodynamic stability. While we do not wish to be bound by theory, itis presently believed that the presence of a 2′-substituent of theinvention results in the sugar moiety of the 2′-substituted nucleosideassuming substantially a 3′-endo conformation and this results in theoligonucleotide-RNA complex assuming an A-type helical conformation.

EXAMPLE 42 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridie(101)

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ML) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 ML) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

EXAMPLE 435′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (102)

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 ML). In the fume hood and withmanual stirring, ethylene glycol (350 ML, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct. NMR (DMSO-d6) d 1.05 (s, 9H, t-butyl), 1.45 (s, 3 H, CH3),3.5-4.1 (m, 8 H, CH2CH2, 3′-H, 4′-H, 5′-H, 5″-H), 4.25 (m, 1 H, 2′-H),4.80 (t, 1 H, CH2O—H), 5.18 (d, 2H, 3′-OH), 5.95 (d, 1 H, 1′-H),7.35-7.75 (m, 11 H, Ph and C6-H), 11.42 (s, 1 H, N—H).

EXAMPLE 442′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(103)

Nucleoside 102 (20 g, 36.98 mmol) was mixed with triphenylphosphine(11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). Itwas then dried over P₂O₅ under high vacuum for two days at 40° C. Thereaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich,sure seal bottle) was added to get a clear solution.Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture. The rate of addition is maintained such that resultingdeep red coloration is just discharged before adding the next drop.After the addition was complete, the reaction was stirred for 4 hrs. Bythat time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residueobtained was placed on a flash column and eluted with ethylacetate:hexane (60:40), to get 103 as white foam (21.819, 86%). Rf 0.56(ethyl acetate:hexane, 60:40). MS (FAB⁻)m/e 684 (M−H⁺)

EXAMPLE 455′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(104)

Compound 103 (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0C.After 1 hr the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eg.) was addedand the mixture for 1 hr. Solvent removed under vacuum; residuechromatographed to get compound 104 as white foam (1.95, 78%). Rf 0.32(5% MeOH in CH₂Cl₂). MS (Electrospray⁻) m/e 566 (M−H^(⊕))

EXAMPLE 465′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(105)

Compound 104 (1.77 g, 3.12 mmol) was dissolved in a solution of 1Mpyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL).Sodiumcyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at10° C. under inert atmosphere. The reaction mixture was stirred for 10minutes at 10° C. After that the reaction vessel was removed from theice bath and stirred at room temperature for 2 hr, the reactionmonitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetatephase dried over anhydrous Na₂SO₄, evaporated to dryness. Residuedissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20%w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred atroom temperature for 10 minutes. Reaction mixture cooled to 10° C. in anice bath, sodiumcyanoborohydride (0.39 g, 6.13 mmol) was added andreaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, thereaction mixture was removed from the ice bath and stirred at roomtemperature for 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL)solution was added and extracted with ethyl acetate (23×25 mL). Ethylacetate layer was dried over anhydrous Na₂SO₄; and evaporated to drynessThe residue obtained was purified by flash column chromatography andeluted with 5% MeOH in CH₂Cl₂ to get 105 as a white foam (14.6 g, 80%).Rf 0.35 (5% MeOH in CH₂Cl₂). MS (FAB^(⊕)) m/e 584 (M+H^(⊕))

EXAMPLE 47 2′-O-(dimethylaminooxyethyl)-5-methyluridine (106)

Triethylamine trihydrofluoride (3.9 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to compound 105 (1.40 g, 2.4mmol) and stirred at room temperature for 24 hrs. Reaction was monitoredby TLC (5% MeOH in CH₂Cl₂). Solvent removed under vacuum and the residueplaced on a flash column and eluted with 10% MeOH in CH₂Cl₂ to get 106(766 mg, 92.5%). Rf 0.27 (5% MeOH in CH₂Cl₂). MS (FAB^(⊕)) m/e 346(M+H^(⊕))

EXAMPLE 48 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (107)

Compound 106 (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuumovernight at 40° C. It was then co-evaporated with anhydrous pyridine(20 mL). The residue obtained was dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to themixture and the reaction mixture was stirred at room temperature untilall of the starting material disappeared. Pyridine was removed undervacuum and the residue chromatographed and eluted with 10% MeOH inCH2Cl2 (containing a few drops of pyridine) to get 107 (1.13 g, 80%). Rf0.44 ((10% MeOH in CH₂Cl₂). MS (FAB^(⊕)) m/e 648 (M+H^(⊕))

EXAMPLE 495′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](108)

Compound 107 (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL).To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) wasadded and dried over P₂O₅ under high vacuum overnight at 40° C. Then thereaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get 108 as a foam (1.04 g, 74.9%). Rf 0.25 (ethylacetate:hexane, 1:1). ³¹P NMR (CDCl₃) δ150.8 ppm; MS (FAB^(⊕)) m/e 848(M+H^(⊕))

EXAMPLE 50 2′/3′-O-allyl adenosine (109)

Adenosine (20 g, 74.84 mmol) was dried over P₂O₅ under high vacuum at40° C. for two days. It was then suspended in DMF under inertatmosphere. Sodium hydride (2.5 g, 74.84 mmol, 60% dispersion in mineraloil), stirred at room temperature for 10 minutes. Then allyl bromide(7.14 mL, 82.45 mmol) was added dropwise and the reaction mixture wasstirred at room temperature overnight. DMF was removed under vacuum andresidue was washed with ethyl acetate (100 mL). Ethyl acetate layer wasdecanted. Filtrate obtained contained product. It was then placed on aflash column and eluted with 10% MeOH in CH₂Cl₂ to get 109 (15.19 g,66%). Rf 0.4, 0.4a ((10% MeOH in CH₂Cl₂)

EXAMPLE 51 2′/3′-O-allyl-N⁶-benzoyl adenosine (110)

Compound 109(15.19 g, 51.1 mmol) was dried over P₂O₅ under high vacuumovernight at 40° C. It was then dissolved in anhydrous pyridine (504.6mL) under inert atmosphere. Trimethylchlorosilane (32.02 mL, 252.3 mmol)was added at 0° C. and the reaction mixture was stirred for 1 hr underinert atmosphere. Then benzoyl chloride (29.4 mL, 252.3 mmol) was addeddropwise. Once the addition of benzoyl chloride was over, the reactionmixture was brought to room temperature and stirred for 4 hrs. Then thereaction mixture was brought to 0° C. in an ice bath. Water (100.9 mL).was added and the reaction mixture was stirred for 30 minutes. ThenNH₄OH (100.0 mL, 30% aqueous solution w/w) was added, keeping thereaction mixture at 0° C. and stirring for an additional 1 hr. Solventevaporated residue partitioned between water and ether. Productprecipitates as an oil, which was then chromatographed (5% MeOH inCH₂Cl₂) to get 13 as a white foam (12.67 g, 62%).

EXAMPLE 52 3′-O-allyl-5′-O-tert-butyldiphenylsilyl-N⁶-benzoyl-adenosine(111)

Compound 110 (11.17 g, 27.84 mmol) was dried over P₂O₅ under vacuum at40° C., then dissolved in dry CH₂Cl₂ (56 mL, sure seal from Aldrich).4-dimethylaminopyridine (0.34 g, 2.8 mmol), triethylamine (23.82 mL, 167mmol) and t-butyldiphenylsilyl chloride were added. The reaction mixturewas stirred vigorously for 12 hr. Reaction was monitored by TLC (ethylacetate:hexane 1:1). It was then diluted with CH₂Cl₂ (50 mL) and washedwith water (3×30 mL) Dichloromethane layer was dried over anhydrousNa₂SO₄ and evaporated to dryness. Residue purified by flashchromatography (ethyl acetate:hexane 1:1 as eluent) to get 111 as awhite foam (8.85 g, 49%). Rf 0.35 (ethyl acetate:hexane, 1:1)

EXAMPLE 535′-O-tert-butyldiphenylsilyl-N⁶-benzoyl-2′-O-(2,3-dihydroxypropyl)-adenosine(112)

Compound 111 (5.5 g, 8.46 mmol), 4-methylmorpholine N-oxide (1.43 g,12.18 mmol) were dissolved in dioxane (45.42 mL). 4% aqueous solution ofOSO₄ (1.99 mL, 0.31 mmol) was added. The reaction mixture was protectedfrom light and stirred for 3 hrs. Reaction was monitored by TLC (5% MeOHin CH₂Cl₂). Ethyl acetate (100 mL) was added and the resulting reactionmixture was washed with water (1×50 mL). Ethyl acetate layer was driedover anhydrous Na₂SO₄ and evaporated to get 112 (5.9 g) and used fornext step without purification. Rf 0.17 (5% MeOH in CH₂Cl₂)

EXAMPLE 545′-O-tert-butyldiphenylsilyl-N⁶-benzoyl-2′-O-(formylmethyl)-adenosine(113)

Compound 112 (5.59 g, 8.17 mmol) was dissolved in dry CH₂Cl₂ (40.42 mL).To this NaIO₄ adsorbed on silica gel (Ref. J. Org. Chem. 1997, 62,2622-2624) (16.34 g, 2 g/mmol) was added and stirred at ambienttemperature for 30 minutes. Reaction monitored by TLC (5% MeOH inCH₂Cl₂). Reaction mixture was filtered and the filtrate washedthoroughly with CH₂Cl₂. Dichloromethane layer evaporated to get thealdehyde 113 (5.60 g) that was used in the next step withoutpurification. Rf 0.3 (5% MeOH in CH₂Cl₂)

EXAMPLE 55 5′-O-tert-butyldiphenylsilyl-N⁶-2′-O-(2-hydroxyethyl)adenosine (114)

Compound 113 (5.55 g, 8.50 mmol) was dissolved in a solution of 1Mpyridinium p-toluenesulfonate in anhydrous MeOH (85 mL). Reactionmixture was protected from moisture. Sodiumcyanoborohydride (1.08 g,17.27 mmol) was added and reaction mixture stirred at ambienttemperature for 5 hrs. The progress of the reaction was monitored by TLC(5% MeOH in CH₂Cl₂). The reaction mixture was diluted with ethyl acetate(150 mL), then washed with 5% NaHCO₃ (75 mL) and brine (75 mL). Ethylacetate layer was dried over anhydrous Na₂SO₄ and evaporated to dryness.Residue purified by flash chromatography (5% MeOH in CH₂Cl₂) to get 114(4.31 g, 77.8%). Rf 0.21 (5% MeOH in CH₂Cl₂). MS (FAB^(⊕)) m/e 655(M+H^(⊕)), 677 (M+Na^(⊕))

EXAMPLE 565′-tert-butyldiphenylsilyl-N⁶-benzoyl-2′-O-(2-phthalimidooxyethyl)adenosine (115)

Compound 114 (3.22 g, 4.92 mmol) was mixed with triphenylphosphine (1.55g, 5.90 mmol) and N-hydroxyphthalimide (0.96 g, 5.90 mmol). It was thendried over P₂O₅ under vacuum at 40° C. for two days. Dissolved driedmixture in anhydrous THF (49.2 mL) under inert atmosphere. Diethylazodicarboxylate (0.93 mL, 5.90 mmol) was added dropwise. The rate ofaddition was maintained such that resulting deep red coloration is justdischarged before adding the next drop. After the addition wascompleted, the reaction was stirred for 4 hrs, monitored by TLC (ethylacetate:hexane 70:30). Solvent was removed under vacuum and the residuedissolved in ethyl acetate (75 mL). The ethyl acetate layer was washedwith water (75 mL), then dried over Na₂SO₄, concentrated andchromatographed (ethylacetate:hexane 70:30) to get 115 (3.60 g, 91.5%).Rf 0.27 ethyl acetate:hexane, 7:3) MS (FAB^(⊕)) m/e 799 (M+H^(⊕)), 821(M+Na^(⊕))

EXAMPLE 575′-O-tert-butyldiphenylsilyl-N⁶-benzoyl-2′-O-(2-formaldoximinooxyethyl)adenosine (116)

Compound 115 (3.5 g, 4.28 mmol) was dissolved in CH₂Cl₂ (43.8 mL).N-methylhydrazine (0.28 mL, 5.27 mmol) was added at −10° C. and thereaction mixture was stirred for 1 hr at −10 to 0° C. Reaction monitoredby TLC (5% MeOH in CH₂Cl₂). A white precipitate formed was filtered andfiltrate washed with ice cold CH₂Cl₂ thoroughly. Dichloromethane layerevaporated on a rotary evaporator keeping the water bath temperature atless than 25° C. Residue obtained was then dissolved in MeOH (65.7 mL).Formaldehyde (710 mL, 4.8 mmol, 20% solution in water) was added and thereaction mixture was stirred at ambient temperature for 1 hr. Reactionmonitored by ¹H NMR. Reaction mixture concentrated and chromatographed(5% MeOH in CH₂Cl₂) to get 116 as a white foam (2.47 g, 83%). Rf 0.37(5% MeOH in CH₂Cl₂). MS (FAB^(⊕)) m/e 681 (M+H^(⊕))

EXAMPLE 585′-tert-butyldiphenylsilyl-N⁶-benzoyl-2′-O-(2-N,N-dimethylaminooxyethyl)adenosine (117)

Compound 116 (2.2 g, 3.23 mmol) was dissolved in a solution of 1Mpyridinium p-toluenesulfonate (PPTS) in MeOH (32 mL). Reaction protectedfrom moisture. Sodium cyanoborohydride (0.31 g) was added at 10° C. andreaction mixture was stirred for 10 minutes at 10° C. It was thenbrought to ambient temperature and stirred for 2 hrs, monitored by TLC(5% MeOH in CH₂Cl₂). 5% aqueous sodium bicarbonate (100 mL) andextracted with ethyl acetate (3×50 mL). Ethyl acetate layer was driedover anhydrous Na₂SO₄ and evaporated to dryness. Residue was dissolvedin a solution of 1M PPTS in MeOH (32 mL). Formaldehyde (0.54 mL, 3.55mmol, 20% aqueous solution) was added and stirred at room temperaturefor 10 minutes. Sodium cyanoborohydride (0.31 g) was added at 10° C. andstirred for 10 minutes at 10° C. Then the reaction mixture was removedfrom ice bath and stirred at room temperature for an additional 2 hrs,monitored by TLC (5% MeOH in CH₂Cl₂). Reaction mixture was diluted with5% aqueous NaHCO₃ (100 mL) and extracted with ethyl acetate (3×50 mL).Ethyl acetate layer was dried, evaporated and chromatographed (5% MeOHin CH₂Cl₂) to get 117 (1.9 g, 81.8%). Rf 0.29 (5% MeOH in CH₂Cl₂). MS(FAB^(⊕)) m/e 697 (M+H^(⊕)), 719 (M+Na^(⊕))

EXAMPLE 59 N⁶-benzoyl-2′-O-(N,N-dimethylaminooxyethyl) adenosine (118)

To a solution of Et₃N-3HF (1.6 g, 10 mmol) in anhydrous THF (10 mL)triethylamine (0.71 mL, 5.12 mmol) was added. Then this mixture wasadded to compound 117 (0.72 g, 1 mmol) and stirred at room temperatureunder inert atmosphere for 24 hrs. Reaction monitored by TLC (10% MeOHin CH₂Cl₂). Solvent removed under vacuum and the residue chromatographed(10% MeOH in CH₂Cl₂) to get 118 (0.409 g, 89%). Rf 0.40 (10% MeOH inCH₂Cl₂). MS (FAB^(⊕)) m/e 459 (M+H^(⊕))

EXAMPLE 605′-O-dimethoxytrityl-N⁶-benzoyl-2′-O-(2-N,N-dimethylaminooxyethyl)adenosine (119)

Compound 118 (0.4 g, 0.87 mmol) was dried over P₂O₅ under vacuumovernight at 40° C. 4-dimethylaminopyridine (0.022 g, 0.17 mmol) wasadded. Then it was co-evaporated with anhydrous pyridine (9 mL). Residuewas dissolved in anhydrous pyridine (2 mL) under inert atmosphere, and4,4′-dimethoxytrityl chloride (0.58 g, 1.72 mmol) was added and stirredat room temperature for 4 hrs. TLC (5% MeOH in CH₂Cl₂) showed thecompletion of the reaction. Pyridine was removed under vacuum, residuedissolved in CH₂Cl₂ (50 mL) and washed with aqueous 5% NaHCO₃ (30 mL)solution followed by brine (30 mL). CH₂Cl₂ layer dried over anhydrousNa₂SO₄ and evaporated. Residue chromatographed (5% MeOH in CH₂Cl₂containing a few drops of pyridine) to get 119 (0.5 g, 75%). Rf 0.20 (5%MeOH in CH₂Cl₂). MS (Electrospray⁻) m/e 759 (M+H^(⊕))

EXAMPLE 61 N⁶-benzoyl-5′-O-DMT-2′-O-(N,N-dimethylaminooxyethyl)adenosine-3′-O-phosphoramidite (120)

Compound 119 (0.47 g, 0.62 mmol) was co-evaporated with toluene (5 mL).Residue was mixed with N,N-diisopropylamine tetrazolide (0.106 g, 0.62mmol) and dried over P₂O₅ under high vacuum overnight. Then it wasdissolved in anhydrous CH₃CN (3.2 mL) under inert atmosphere.2-cyanoethyl-tetraisopropyl phosphordiamidite (0.79 mL, 2.48 mmol) wasadded dropwise and the reaction mixture was stirred at room temperatureunder inert atmosphere for 6 hrs. Reaction was monitored by TLC (ethylacetate containing a few drops of pyridine). Solvent was removed, thenresidue was dissolved in ethyl acetate (50 mL) and washed with 5%aqueous NaHCO₃ (2×25 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄, evaporated, and residue chromatographed (ethyl acetatecontaining a few drops of pyridine) to get 120 (0.45 g, 76%). MS(Electrospray⁻) m/e 959 (M+H^(⊕)) ³¹P NMR (CDCl₃) δ151.36, 150.77 ppm

EXAMPLE 62 2′/3′-O-allyl-2,6-diaminopurine riboside (121 and 122)

2,6-Diaminopurine riboside (30 g, 106.4 mmol) was suspended in anhydrousDMF (540 ML). Reaction vessel was flushed with argon. Sodium hydride(3.6 g, 106.4 mmol, 60% dispersion in mineral oil) was added and thereaction stirred for 10 min. Allyl bromide (14.14 mL, 117.22 mmol) wasadded dropwise over 20 min. The resulting reaction mixture stirred atroom temperature for 20 hr. TLC (10% MeOH in CH₂Cl₂) showed completedisappearance of starting material. DMF was removed under vacuum and theresidue absorbed on silica was placed on a flash column and eluted with10% MeOH in CH₂Cl₂. Fractions containing mixture of 2′ and 3′ allylatedproduct was pooled together and concentrated to dryness to yield amixture of 121 and 122 (26.38 g, 77%). Rf 0.26, 0.4 (10% MeOH in CH₂Cl₂)

EXAMPLE 63 2′-O-allyl-guanosine (123)

A mixture of 121 and 122 (20 g, 62.12 mmol) was suspended in 100 mmsodium phosphate buffer (pH 7.5) and adenosine deaminase (1 g) wasadded. The resulting solution was stirred very slowly for 60 hr, keepingthe reaction vessel open to atmosphere. Reaction mixture was then cooledin ice bath for one hr and the precipitate obtained was filtered, driedover P₂O₅ under high vacuum to yield 123 as white powder (13.92 g, 69.6%yield). Rf 0.19 (20% MeOH in CH₂Cl₂)

EXAMPLE 64 2′-O-allyl-3′, 5′-bis(tert-butyl diphenylsilyl) guanosine(124)

2′-O-allyl-guanosine (6 g, 18.69 mmol) was mixed with imidazole (10.18g, 14.952 mmol) and was dried over P₂O₅ under high vacuum overnight. Itwas then flushed with argon. Anhydrous DMF (50 mL) was added and stirredwith the reaction mixture for 10 minutes. To thistert-butyldiphenylsilyl chloride (19.44 mL, 74.76 mmol) was added andthe reaction mixture stirred overnight under argon atmosphere. DMF wasremoved under vacuum and the residue was dissolved in ethyl acetate (100mL) and washed with water (2×75 mL). Ethyl acetate layer was dried overanhydrous Na₂SO₄ and evaporated to dryness. Residue placed on a flashcolumn and eluted with 5% MeOH in CH₂Cl₂. Fractions containing theproduct were pooled together and evaporated to yield 124 (10.84 g, 72%yield) as a white foam. Rf=? MS (FAB^(⊕)) m/e 800 (M+H^(⊕)), 822(M+Na^(⊕))

EXAMPLE 65 2′-O-(2-hydroxyethyl)-3′, 5′-bis(tert-butyldiphenylsilyl)guanosine (125)

Compound 124 (9 g, 11.23 mmol) was dissolved in CH₂Cl₂ (80 mL). To theclear solution acetone (50 ML), 4-methyl morpholine-N-oxide (1.89 g,16.17 mmol) was added. The reaction flask was protected from light. Thus4% aqueous solution of osmium tetroxide was added and the reactionmixture was stirred at room temperature for 6 hr. Reaction volume wasconcentrated to half and ethyl acetate (50 mL) was added. It was thenwashed with water (30 mL) and brine (30 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. Residue was thendissolved in CH₂Cl₂ and NaIO₄ adsorbed on silica (21.17 g, 2 g/mmol) wasadded and stirred with the reaction mixture for 30 min. The reactionmixture was filtered and silica was washed thoroughly with CH₂Cl₂.Combined CH₂Cl₂ layer was evaporated to dryness. Residue was thendissolved in dissolved in 1M pyridinium-p-toluene sulfonate (PPTS) indry MeOH (99.5 mL) under inert atmosphere. To the clear solution sodiumcyanoborohydride (1.14 g, 18.2 mmol) was added and stirred at roomtemperature for 4 hr. 5% aqueous sodium bicarbonate (50 mL) was added tothe reaction mixture slowly and extracted with ethyl acetate (2×50 mL).Ethyl acetate layer was dried over anhydrous Na₂SO₄ and evaporated todryness. Residue placed on a flash column and eluted with 10% MeOH inCH₂Cl₂ to yield 125 (6.46 g, 72% yield). MS (Electrospray−) m/e 802(M−H^(⊕))

EXAMPLE 66 2′-O-[(2-phthalimidoxy)ethyl]-3′, 5′-bis(tertbutyldiphenylsilyl) guanosine (126)

Compound 125 (3.7 g, 4.61 mmol) was mixed with Ph₃P (1.40 g, 5.35 mmol),and hydroxy phthalimide (0.87 g, 5.35 mmol). It was then dried over P₂O₅under high vacuum for two days at 40° C. These anhydrous THF (46.1 mmol)was added to get a clear solution under inert atmosphere.Diethylazidocarboxylate (0.73 mL, 4.61 mmol) was added dropwise in sucha manner that red color disappears before addition of the next drop.Resulting solution was then stirred at room temperature for 4 hr. THFwas removed under vacuum and the residue dissolved in ethyl acetate (75mL) and washed with water (2×50 mL). Ethyl acetate layer was dried overanhydrous Na₂SO₄ and concentrated to dryness. Residue was purified bycolumn chromatography and eluted with 7% MeOH in ethyl acetate to yield126 (2.62 g, 60% yield). Rf 0.48 (10% MeOH in CH₂Cl₂). MS (FAB⁻) m/e 947(M−H^(⊕)).

EXAMPLE 67 2′-O-(2-phthalimido-N-oxyethyl)-3′,5′-O-bis-tert-butyldiphenylsilyl-N2-isobutyrylguanosine (127)

2′-O-(2-phthalimido-N-oxyethyl)-3′, 5′-O-bis-tert-butyldiphenylsilylguanosine (3.66 g, 3.86 mmol) was dissolved in anhydrous pyridine (40ML), the solution was cooled to 5° C., and isobutyryl chloride (0.808ML, 7.72 mmol) was added dropwise. The reaction mixture was allowed towarm to 25° C., and after 2 h additional isobutyryl chloride (0.40 ML,3.35 mmol) was added at 25° C. After 1 h the solvent was evaporated invaccuo (0.1 torr) at 30° C. to give a foam which was dissolved in ethylacetate (150 ML) to give a fine suspension. The suspension was washedwith water (2×15 ML) and brine (4 ML), and the organic layer wasseparated and dried over MgSO₄. The solvent was evaporated in vaccuo togive a foam, which was purified by column chromatography usingCH₂Cl₂—MeOH, 94:6, v/v, to afford the title compound as a white foam(2.57 g, 65%). ¹H NMR(CDCl₃): d 11.97 (br s, 1H), 8.73 (s, 1H), 7.8-7.2(m, 25H), 5.93 (d, 1H, J_(1′,2′)=3.3 Hz), 4.46 (m, 1H), 4.24 (m, 2H),3.83 (m, 2H), 3.60 (m, 2H), 3.32 (m, 1H), 2.67 (m, 1H), 1.30 (d, 3H,J=3.2 Hz), 1.26 (d, 3H, j=3.1 Hz), 1.05 (s, 9H), 1.02 (s, 9H).

This compound was further derivatized into the correspondingphosphoramidite using the chemistries described above for A and Tanalogs to give compound 128.

EXAMPLE 683′-O-acetyl-2′-O-(2-N,N-dimethylaminooxyethyl)-5′-O-tert-butyldiphenylsilylthymidine (129)

Compound 105 (3.04 g, 5.21 mmol) was dissolved in chloroform (11.4 mL).To this was added dimethylaminopyridine (0.99 g, 8.10 mmol) and thereaction mixture was stirred for 10 minutes. Acetic anhydride (0.701 g,6.87 mmol) was added and the reaction mixture was stirred overnight. Thereaction mixture was then diluted with CH₂Cl₂ (40 mL) and washed withsaturated NaHCO₃ (30 ML) and brine (30 ML). CH₂Cl₂ layer evaporated todryness. Residue placed on a flash column and eluted with ethylacetate:hexane (80:20) to yield 129. Rf 0.43 (ethyl acetate:hexane,80:20). MS (Electrospray⁻) m/e 624 (M−H⁻)

EXAMPLE 692′-O-(2-N,N-dimethylaminooxyethyl)-5′-O-tert-butyldiphenylsilyl 5-methylcytidine (130)

A suspension of 1,2,4-triazole (5.86 g, 84.83 mmol) in anhydrous CH₃CN(49 mL) was cooled in an ice bath for 5 to 10 min. under argonatmosphere. To this cold suspension POCl₃ (1.87 mL, 20 mmol) was addedslowly over 10 min. and stirring continued for an additional 5 min.Triethylamine (13.91 mL, 99.8 mmol) was added slowly over 30 min.,keeping the bath temperature around 0-2° C. After the addition wascomplete the reaction mixture was stirred at this temperature for anadditional 30 minutes when compound 35 (3.12 g, 4.99 mmol) was added inanhydrous acetonitrile (3 mL) in one portion. The reaction mixture wasstirred at 0-2° C. for 10 min. Then ice bath was removed and thereaction mixture was stirred at room temperature for 1.5 hr. Thereaction mixture was cooled to ° C. and this was concentrated to smallervolume and dissolved in ethyl acetate (100 mL), washed with water (2×30mL) and brine (30 mL). Organic layer was dried over anhydrous Na₂SO₄ andconcentrated to dryness. Residue obtained was then dissolved insaturated solution of NH₃ in dioxane (25 mL) and stirred at roomtemperature overnight. Solvent was removed under vacuum. The residue waspurified by column chromatography and eluted with 10% MeOH in CH₂Cl₂ toget 130.

EXAMPLE 702′-O-(2,N,N-dimethylaminooxyethyl)-N⁴-benzoyl-5′-O-tert-butyldiphenylsilylcytidine(131)

Compound 130 (2.8 g, 4.81 mmol) was dissolved in anhydrous DMF (12.33mL). Benzoic anhydride (1.4 g, 6.17 mmol) was added and the reactionmixture was stirred at room temperature overnight. Methanol was added (1mL) and solvent evaporated to dryness. Residue was dissolved indichloromethane (50 mL) and washed with saturated solution of NaHCO₃(2×30 ML) followed by brine (30 mL). Dichloromethane layer was driedover anhydrous Na₂SO₄ and concentrated. The residue obtained waspurified by column chromatography and eluted with 5% MeOH in CH₂Cl₂ toyield 131 as a foam.

EXAMPLE 71 N⁴-Benzoyl-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methylcytidine (132)

Compound 131 (2.5 g, 3.9 mmol) was dried over P₂O₅ under high vacuum. Ina 100 mL round bottom flask, triethylamine trihydrofluoride (6.36 mL, 39mmol) is dissolved in anhydrous THF (39 mL). To this, triethylamine(2.72 mL, 19.5 mmol) was added and the mixture was quickly poured intocompound 131 and stirred at room temperature overnight. Solvent isremoved under vacuum and the residue kept in a flash column and elutedwith 10% MeOH in CH₂Cl₂ to yield 132.

EXAMPLE 72N⁴-Benzoyl-2′-O-(2-N,N-dimethylaminooxyethyl)-5-O′-dimetoxytrityl-5-methylcytidine (133)

Compound 132 (1.3 g, 2.98 mmol) was dried over P₂O₅ under high vacuumovernight. It was then co-evaporated with anhydrous pyridine (10 mL).Residue was dissolved in anhydrous pyridine (15 mL), 4-dimethylaminopyridine (10.9 mg, 0.3 mmol) was added and the solution was stirred atroom temperature under argon atmosphere for 4 hr. Pyridine was removedunder vacuum and the residue dissolved in ethyl acetate and washed with5% NaHCO₃ (20 mL) and brine (20 mL). Ethyl acetate layer was dried overanhydrous Na₂SO₄ and concentrated to dryness. Residue was placed on aflash column and eluted with 10% MeOH in CH₂Cl₂ containing a few dropsof pyridine to yield compound 133.

EXAMPLE 73N⁴-Benzoyl-2′-O-(2-N,N-dimethylaminooxyethyl)-5-dimethoxytrityl-5-methylcytidine-3′-O-phosphoramidite (134)

Compound 133 (1.54 g, 2.09 mmol) was co-evaporated with toluene (10 mL).It was then mixed with diisopropylamine tetrazolide (0.36 g, 2.09 mmol)and dried over P₂O₅ under high vacuum at 40° C. overnight. Then it wasdissolved in anhydrous acetonitrile (11 mL) and2-cyanoethyl-tetraisopropylphosphoramidite (2.66 mL, 8.36 mmol) wasadded. The reaction mixture was stirred at room temperature under inertatmosphere for 4 hr. Solvent was removed under vacuum. Ethyl acetate (50mL) was added to the residue and washed with 5% NaHCO₃ (30 mL) and brine(30 mL). Organic phase was dried over anhydrous Na₂SO₄ and concentratedto dryness. Residue placed on a flash column and eluted withethylacetate:hexane (60:40) containing a few drops of pyridine to get134.

EXAMPLE 74 2′-O-dimethylaminooxyethyl-2,6-diaminopurine ribosidephosphoramidite (135)

For the incorporation of 2′-O-dimethylaminooxyethyl-2,6-diaminopurineriboside into oligonucleotides, we elected to use the phosphoramidite ofprotected 6-amino-2-fluoropurine riboside 135. Post-oligo synthesis,concomitant with the deprotection of oligonucleotide protection groups,the 2-fluoro group is displaced with ammonia to give the2,6-diaminopurine riboside analog. Thus, 2,6-diaminopurine riboside isalkylated with dimethylaminooxyethylbromide 136 to afford a mixture of2′-O-dimethylaminooxyethyl-2,6-diaminopurine riboside 137 and the3′-isomer 138. Typically after functionalizing the 5′-hydroxyl with DMTto provide5′-O-(4,4′-dimethoxytrityl)-2′-O-dimethylaminooxyethyl-2,6-diaminopurineriboside 139, the 2′-isomer may be resolved chromatographically.Fluorination of 139 via the Schiemann reaction (Krolikiewicz, K.;Vorbruggen, H. Nucleosides Nucleotides, 1994, 13, 673-678) provides2′-O-dimethylaminooxyethyl-6-amino-2-fluoro-purine riboside 140 andstandard protection protocols affords5′-O-(4,4′-dimethoxytrityl)-2′-O-dimethylaminooxyethyl-6-dimethyformamidine-2-fluoropurineriboside 140. Phosphitylation of 140 gives5′-O-(4,4′-dimethoxytrityl)-2′-O-dimethylaminooxyethyl-6-dimethyformamidine-2-fluoropurineriboside-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 138.

In the event that compound 139 cannot be resolved chromatographicallyfrom the 3′-isomer, the mixture of compounds 137 and 138 may be treatedwith adenosine deaminase, which is known to selectively deaminate2′-O-substituted adenosine analogs in preference to the 3′-O-isomer, toafford 2′-O-dimethylaminooxyethylguanosine 142.5′-O-(4,4′-dimethoxytrityl)-2′-O-dimethylaminooxyethylguanosine 140 maybe converted to the 2,6-diaminopurine riboside analog 139 by aminationof the 6-oxo group (Gryaznov, S.; Schultz, R. G. Tetrahedron Lett. 1994,2489-2492). This was then converted to the corresponding amidite 144 bystandard protection methods and protocols for phosphitylation.

EXAMPLE 752′/3′-O-[2-(tert-butyldimethylsilylhydroxy)ethyl]-2,6-diaminopurineriboside (145 and 146)

2,6-diaminopurine riboside (10 g, 35.46 mmol) was dried over P₂O₅ underhigh vacuum. It was suspended in anhydrous DMF (180 mL) and NaH (1.2 g,35.46 mmol, 60% dispersion in mineral oil) was added. The reactionmixture was stirred at ambient temperature at inert atmosphere for 30minutes. To this (2-bromoethoxy)-tert-butyldimethylsilane (12.73 g, 53.2mmol) was added dropwise and the resulting solution was stirred at roomtemperature overnight. DMF was removed under vacuum, residue wasdissolved in ethyl acetate (100 mL) and washed with water (2×70 mL).Ethyl acetate layer was dried over anhydrous MgSO₄ and concentrated todryness. Residue was placed on a flash column and eluted with 5% MeOH inCH₂Cl₂ to get a mixture of products (6.0711 g, 31% yield). Rf 0.49,0.59, 0.68 (5% MeOH in CH₂Cl₂).

EXAMPLE 76 2′-O-aminooxyethyl analogs

Various other 2′-O-aminooxyethyl analogs of nucleoside (for e.g.,2,6-diaminopurine riboside) may be prepared as compounds 154. Thus,alkylation of 2,6-diamino purine with(2-bromoethoxy)-tert-butyldimethylsilane gives2′-O-tert-butyldimethylsilyloxyethyl-2,6-diaminopurine riboside 145 andthe 3′-isomer 146. The desired 2′-O-isomer may be resolved bypreparation of5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilyloxyethyl-2,6-diaminopurineriboside 147 and subjecting the mixture to column chromatography.Deprotection of the silyl group provides5′-O-(4,4′-dimethoxytrityl)-2′-O-hydroxyethyl-2,6-diaminopurine riboside148 which undergoes a Mitsunobu reaction to give5′-O-(4,4′-dimethoxytrityl)-2′-O-(2-phthalimido-N-oxyethyl)-2,6-diaminopurineriboside 149. Treatment of 149 under Schiemann conditions effectsfluorination and deprotection of the DMT group to yield2′-O-(2-phthalimido-N-oxyethyl)-6-amino-2-fluoropurine riboside 150.Standard protection conditions provides5′-O-(4,4′-dimethoxytrityl)-2′-O-(2-phthalimido-N-oxyethyl)-6-dimethyformamidine-2-fluoropurineriboside 151 and deprotection of the phthalimido function affords5′-O-(4,4′-dimethoxytrityl)-2′-O-aminooxyethyl-6-dimethyformamidine-2-fluoropurineriboside 152.

Reductive amination of 152 with aldehydes or dialdehydes results incyclic or acyclic disubstituted 2′-O-aminooxyethyl analogs 153.Phosphitylation of 153 provides cyclic or acyclic disubstituted2′-O-aminooxyethyl analogs 154 as the phosphoramidites.

EXAMPLE 77 2′/3′-O(2-tert-butyldimethylsilylhydroxyethyl)adenosine (155and 156)

Adenosine (10 g, 37.42 mmol) was dried over P₂O₅ under high vacuum. Itwas then suspended in anhydrous DMF (150 ML) and NaH (1.35 g, 56.13mmol) was added. The reaction mixture was stirred at room temperatureunder inert atmosphere for 30 min. Then (2-bromoethyl)-tert-butyldimethylsilane (9.68 mL, 4.4.90 mmol) was addeddropwise and the reaction mixture stirred at room temperature overnight.DMF was removed under vacuum and to the residue dichloromethane (100 mL)was added and washed with water (2×80 mL). Dichloromethane layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. Residue purifiedby column to get a mixture of products (4.30 g). Rf 0.49, 0.57 (10% MeOHin CH₂Cl₂)

EXAMPLE 78 2′-O-(2-methyleneiminooxyethyl)thymidine (157)

Compound 104 (3.10 g, 5.48 mmol) was dried over P₂O₅ under high vacuum.In a 100 mL round bottom flask, triethylamine-trihydroflouride (8.93 mL,54.8 mmol) was dissolved in anhydrous THF and triethylamine (3.82 mL,27.4 mmol) was added. The resulting solution was immediately added tothe compound 104 and the reaction mixture was stirred at roomtemperature overnight. Solvent was removed under vacuum. Residueobtained was placed on a flash column and eluted with 10% MeOH in CH₂Cl₂to yield 157 as white foam (1.35 g, 75% yield). Rf 0.45 (5% MeOH inCH₂Cl₂). MS (FAB^(⊕)) m/e 330 (M+H^(⊕)), 352 (M+Na^(⊕)).

EXAMPLE 79 5′-O-dimethoxytrityl-2′-O-(2-methyleneiminooxyethyl)thymidine(158)

Compound 157 (0.64 g, 1.95 mmol) was dried over P₂O₅ under high vacuumovernight. It was then co-evaporated with anhydrous pyridine (5 mL).Residue dissolved in anhydrous pyridine (4.43 mL) and dimethoxytritylchloride (0.79 g, 2.34 mmol), and 4-dimethylaminopyridine (23.8 mg, 0.2mmol) was added. Reaction mixture was stirred under inert atmosphere atambient temperature for 4 hrs. Solvent was removed under vacuum, theresidue purified by column and eluted with 5% MeOH in CH₂Cl₂ containinga few drops of pyridine to yield 158 as a foam (1.09 g, 88% yield). Rf0.4 (5% MeOH in CH₂Cl₂). MS (Electrospray⁻) m/e 630 (M−H^(⊕))

EXAMPLE 805′-O-dimethoxytrityl-2′-O-(2-methyleneiminooxyethyl)thymidine-3′-O-phosphoramidite(159)

Compound 158 (0.87 g, 1.34 mmol) was co-evaporated with toluene (10 mL).Residue was then mixed with diisopropylamine tetrazolide (0.23 g, 1.34mmol) and dried over P₂O₃ under high vacuum overnight. It was thenflushed with argon. Anhydrous acetonitrile (6.7 mL) was added to get aclear solution. To this solution 2-cyanoethyltetraisopropylphosphorodiamidites (1.7 mL, 5.36 mmol) was added and thereaction mixture was stirred at room temperature for 6 hr. under inertatmosphere. Solvent was removed under vacuum, the residue was dilutedwith ethyl acetate (40 mL), and washed with 5% NaHCO₃ (20 mL) and brine(20 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ andconcentrated to dryness. Residue placed on a flash column and elutedwith ethyl acetate:hexane (60:40) to yield 159 (1.92 g, 80% yield). Rf0.34 (ethyl acetate:hexane, 60:40). ³¹P NMR (CDCl₃) δ150.76 ppm, MS(Electrospray⁻) m/e 830 (M−H^(⊕)).

EXAMPLE 81 Attachment of Nucleoside to Solid Support General Procedure

Compound 107 (200 mg, 0.31 mmol) was mixed with DMAP (19 mg, 16 mmol),succinic anhydride (47 mg, 0.47 mmol), triethylamine (86 mL, 0.62 mmol)and dichloromethane (0.8 mL) and stirred for 4 hr. The mixture wasdiluted with CH₂Cl₂ (50 mL) and the CH₂Cl₂ layer was washed first withice cold 10% aqueous citric acid and then with water. The organic phasewas concentrated to dryness to yield 161. Residue (161) was dissolved inanhydrous acetonitrile (23 mL). To this DMAP (37 mg, 0.3 mmol), and2′,2′-dithiobis(5-nitropyridine) (103 mg, 0.33 mmol) were added. Thesolution was stirred for 5 min. To this was added triphenylphosphine(78.69 mg, 0.3 mmol) in anhydrous acetonitrile (3 mL). The solution wasstirred for 10 min. and then CPG was added to it. The slurry was thenshaken for 2 hr. It was then filtered, washed with acetonitrile andCH₂Cl₂. The functionalized CPG was dried and capped with cappingsolution to yield 161. Loading capacity was determined (58.3 μmol/g).

EXAMPLE 82 Synthesis of Aminooxy Derivatives: Alternative Procedure

The diol 162 is converted to its tosylate derivative 163 by treatmentwith 1 equivalent of p-toluenesulfonyl chloride-pyridine followed bystandard work-up. The tosylate is subsequently treated with severalamino-hydroxy compounds to act as nucleophiles in displacing tosylate toyield a series of oxy-amino compounds. The reaction is facilitated bypreforming the anion from the amino alcohol or hydroxylamine derivativeby the use of sodium hydride under anhydrous conditions.

EXAMPLE 83 General Procedure for the Preparation of DMAOEOligonucleotides and Gapped Oligonucleotides

A 0.1 m solution of each 2′-O-DMAOE amidite was prepared as a solutionin anhydrous acetonitrile and loaded onto an Expedite Nucleic Acidsynthesis system (Millipore) to synthesize oligonucleotides. All otheramidites (A, T, C and G, PerSeptive Biosystem) used in synthesis alsomade as 0.1 M solution in anhydrous acetonitrile. All syntheses werecarried out in the DMT on mode. For the coupling of the 2′-O-DMAOEamidites coupling time was extended to 10 minutes and this step wascarried out twice. All other steps in the protocol supplied by Milliporewere used except the extended oxidation time (240 seconds). 0.5 msolution of (S)-(+)-10-camphorsulfoyl)oxaziridine in anhydrousacetonitrile was used as oxidizer. Beaucage reagent was used forphosphorothioate synthesis. The overall coupling efficiencies were morethan 90%. The oligonucleotides were cleaved from the controlled poreglass (CPG) supports and deprotected under standard conditions usingconcentrated aqueous NH₄OH (30%) at 55° C. 5′-O-DMT containing oligomerswere then purified by reverse phase liquid chromatography (C-4, Waters,7-8×300 mm, A=50 mM triethylammonium acetate pH 1, B=100% CH₃CN, 5 to60% B in 60 minutes). Detritylation with aqueous 80% acetic acid (1 ML,30 min., room temperature), evaporations, followed by desalting by usingsephadese G-25 column gave oligonucleotides as pure foams. All oligomerswere then analyzed by CGE, HPLC and mass spectrometry.

DMAOE GAPMERS HPLC Reten- SEQ Mass tion ID Exp. Obs. time Target NO:Sequence 5′-3′ g/mol g/mol minutes Target 20 T*T*C*T* C*GCCCG 7440.687439.55 23.62 c-raf CTC* T*C*C*T*C*C* 19 T*T*C*T* C*GCTGGT 7018.437017.69 23.90 pkc-α GAGT* T*T*C*A* 20 T*T*C*T*C*GCCCGC 7600.68 7601.1225.60 c-raf TCC*T*C*C*T*C*C* 19 T*T*C*T*C*G CTGGT 7146.44 7146.44 25.91pkc-α GAGT*T*T*C*A* 20 T*T*C*T*C*GCCCGC 7543.30 7541.90 24.83 c-rafTCC*T*C*C*T*C*C* 19 T*T*C*T*C*GCTGGT 7189.71 7188.41 25.28 pkc-αGAGT*T*T*C*A* 20 T*T*C*T*C*GCCCGCT 7383.30 7379.64 22.60 c-rafCC*T*C*C*T*C*C* 19 T*T*C*T*C*GCTGGTG 7061.71 7059.70 23.02 pkc-αAGT*T*T*C*A*

are modified as 2′-O-MOE and SEQ ID NoS., 19 and 20 are modified as2′-O-DMAOE; underlined nucleotides are joined by phosphorothioatelinkages and all other internucleotide linkages are phosphodiester; allC's are 5-methyl C; and separations were performed using the followingHPLC conditions: C-4 column, Waters 3.9×300 m.m, A=50 mM TEAAc, B=CH₃CN,5 to 60% in 60 min. Flow 1.5 ML/min., t=260 nm.

For the synthesis of the foregoing oligonucleotides, especially the MOEgapmers, as controls the following modified amidites were used:2′-O-methoxyethyl-thymidine (RIC,Inc. lot # E1050-P-10),2′-O-methoxyethyl-5-methylcytidine (lot # S1941/RS ),2′-O-methoxyethyl-adenosine, and 5-methylcytidine (lot # 311094).

The required amounts of the amidites were placed in dried vials,dissolved in acetonitrile (unmodified nucleosides were made into 1Msolutions and modified nucleosides were 100 mg/ML), and connected to theappropriate ports on a Millipore Expedite™ Nucleic Acid Synthesis System(ISIS 4). 30 mg of solid support resin was used in each column for 1umole scale synthesis. The synthesis was run using the IBP-PS(1umole)decoupling protocol for phosphorothioate backbones and CSO-8 forphosphodiesters. The trityl reports indicated normal coupling results.

After synthesis the oligonucleotides were deprotected with conc.ammonium hydroxide(aq) at 55° C. for approximately 16 hrs. Then theywere evaporated, using a Savant AS160 Automatic SpeedVac, (to removeammonia) and filtered to remove the CPG-resin.

The crude samples were analyzed by MS, HPLC, and CE. Then they werepurified on a Waters 600E HPLC system with a 991 detector using a WatersC4 Prep. scale column (Alice C4 Prep. Oct. 16, 1996) and the followingsolvents: A: 50 mM TEA-Ac, pH 7.0 and B: acetonitrile utilizing the“MPREP2” method.

After purification the oligos were evaporated to dryness and thendetritylated with 80% acetic acid at room temp. for approximately 30min. Then they were evaporated.

The oligos were then dissolved in conc. ammonium hydroxide and runthrough a column containing Sephadex G-25 using water as the solvent anda Pharmacia LKB SuperFrac fraction collector. The resulting purifiedoligos were evaporated and analyzed by MS, CE, and HPLC.

EXAMPLE 83 General Procedure for the Preparation of DMAOEOligonucleotides and Gapped Oligonucleotides

A 0.1 m solution of each 2′-O-DMAOE amidite was prepared as a solutionin anhydrous acetonitrile and loaded onto an Expedite Nucleic Acidsynthesis system (Millipore) to synthesize oligonucleotides. All otheramidites (A, T, C and G, PerSeptive Biosystem) used in synthesis alsomade as 0.1 M solution in anhydrous acetonitrile. All syntheses werecarried out in the DMT on mode. For the coupling of the 2′-O-DMAOEamidites coupling time was extended to 10 minutes and this step wascarried out twice. All other steps in the protocol supplied by Milliporewere used except the extended oxidation time (240 seconds). 0.5 msolution of (S)-(+)-10-camphorsulfoyl)oxaziridine in anhydrousacetonitrile was used as oxidizer. Beaucage reagent was used forphosphorothioate synthesis. The overall coupling efficiencies were morethan 90%. The oligonucleotides were cleaved from the controlled poreglass (CPG) supports and deprotected under standard conditions usingconcentrated aqueous NH₄OH (30%) at 55° C. 5′-O-DMT containing oligomerswere then purified by reverse phase liquid chromatography (C-4, Waters,7-8×300 mm, A=50 mM triethylammonium acetate pH 1, B=100%CH₃CN, 5 to 60%B in 60 minutes). Detritylation with aqueous 80% acetic acid (1 ML, 30min., room temperature), evaporations, followed by desalting by usingsephadese G-25 column gave oligonucleotides as pure foams. All oligomerswere then analyzed by CGE, HPLC and mass spectrometry.

DMAOE GAPMERS HPLC Reten- SEQ Mass tion ID Exp. Obs. time Target NO:Sequence 5′-3′ g/mol g/mol minutes Target 20a T*T*C* T*C*G CCC 7440.687439.55 23.62 c-raf GCT* CCT* C*C*T* C*C* 19a T*T*C* T*C*G CTG 7018.437017.69 23.90 pkc-α GTG AGT* T*T*C* A* 20a T_(S)*T_(S)*C_(S)*T_(S)*C_(S)*G 7600.68 7601.12 25.60 c-raf CCC GCT CC*T_(S)*C_(S)*C_(S)*T_(S)* C_(S)*C* 19a T_(S)*T_(S)*C_(S)* T_(S)*C_(S)*G 7146.447146.44 25.91 pkc-α CTG GTG AGT* T*T*C* A* 20b T_(S)*T_(S)*C_(S)*T_(S)*C_(S)*G 7543.30 7541.90 24.83 c-raf CCC GCT CC*T_(S)*C_(S)*C_(S)*T_(S)* C_(S)*C* 19b T_(S)*T_(S)*C_(S)* T_(S)*C_(S)*G 7189.717188.41 25.28 pkc-α CTG GTG AGT_(S)* T_(S)*T_(S)*C_(S)* A_(S)* 20bT*T*C* T*C*G CCC 7383.30 7379.64 22.60 c-raf GCT CC*T* C*C*T* C*C* 19bT*T*C* T*C*G CTG 7061.71 7059.70 23.02 pkc-α GTG AGT* T*T*C* A*

modified as 2′-O-MOE and 19b and 20b are modified as 2′-O-DMAOE;subscript s indicates a phosphorothioate internucleoside linkage and allother internucleotide linkages are phosphodiester; all C's are 5-methylC; and separations were performed using the following HPLC conditions:C-4 column, Waters 3.9×300 m.m, A=50 mM TEAAc, B=CH₃CN, 5 to 60% in 60min. Flow 1.5 ML/min., t=260 nm.

For the synthesis of the foregoing oligonucleotides, especially the MOEgapmers, as controls the following modified amidites were used:2′-O-methoxyethyl-thymidine (RIC,Inc. lot # E1050-P-10),2′-O-methoxyethyl-5-methylcytidine (lot # S1941/RS),2′-O-methoxyethyl-adenosine, and 5-methylcytidine (lot # 311094).

The required amounts of the amidites were placed in dried vials,dissolved in acetonitrile (unmodified nucleosides were made into 1Msolutions and modified nucleosides were 100 mg/ML), and connected to theappropriate ports on a Millipore Expedite™ Nucleic Acid Synthesis System(ISIS 4). 30 mg of solid support resin was used in each column for 1umole scale synthesis. The synthesis was run using the IBP-PS(1umole)decoupling protocol for phosphorothioate backbones and CSO-8 forphosphodiesters. The trityl reports indicated normal coupling results.

After synthesis the oligonucleotides were deprotected with conc.ammonium hydroxide(aq) at 55° C. for approximately 16 hrs. Then theywere evaporated, using a Savant AS160 Automatic SpeedVac, (to removeammonia) and filtered to remove the CPG-resin.

The crude samples were analyzed by MS, HPLC, and CE. Then they werepurified on a Waters 600E HPLC system with a 991 detector using a WatersC4 Prep. scale column (Alice C4 Prep. Oct. 16, 1996) and the followingsolvents: A: 50 mM TEA-Ac, pH 7.0 and B: acetonitrile utilizing the“MPREP2” method.

After purification the oligos were evaporated to dryness and thendetritylated with 80% acetic acid at room temp. for approximately 30min. Then they were evaporated.

The oligos were then dissolved in conc. ammonium hydroxide and runthrough a column containing Sephadex G-25 using water as the solvent anda Pharmacia LKB SuperFrac fraction collector. The resulting purifiedoligos were evaporated and analyzed by MS, CE, and HPLC. These oligomersare the 2′-DMAOE thioate and diester analogs of SEQ ID NOs. 19 and 20.

SEQ ID Mass (g/mol) Mass (g/mol) NO: Observed Observed 20 7240.9297239.91 (P = S wings) 19 6887.341 6882.51 (P = S wings) 20 7080.9297076.04 (P = O wings) 19 6759.341 6756.51 (P = O wings)

EXAMPLE 84 General Procedure for the Preparation of Uniformly ModifiedDMAOE Oligonucleotides

2-O-DMAOE amidites of A (225 mg, 0.23 mmol), ^(5me)C (150 mg,0.16 mmol),G (300 mg, 0.31 mmol) and T (169.4 mg, 0.2 mmol) were dissolved inanhydous acetonitrile to get 0.1 M solutions. These solutions wereloaded onto a Expedite Nucleic Acid Synthesis system (Millipore) tosynthesize the oligonucleotides. The coupling efficiencies were morethan 90%. For the coupling of the amidite 1 coupling time was extendedto 10 minutes and this step was carried out twice. All other steps inthe protocol supplied by Millipore were used except the extendedcoupling time. Because reagent (0.1 M in acetonitrile) was used as asulferizing agent. For diester synthesis, CSO was used as the oxidizingagent.

The oligomers were cleaved from the controlled pore glass(CPG) supportsand deprotected under standard conditions using concentrated aqueousNH₄OH (30%) at 55 ° C. 5′-O-DMT containing oligomers were then purifiedby reverse phase high performance liquid chromatography (C-4, Waters,7.8×300 mm, A=50 mM triethylammonium acetate, pH −7, B=acetonitrile,5-60% of B in 60 min., flow 1.5 ML/min.). Detritylation with aqueous 80%acetic acid and evaporation, followed by desalting in a Sephadex G-25column gave oligonucleotides 28059, 28060 and 22786. Oligonucleotideswere analyzed by HPLC, CGE and Mass spectrometry.

SEQ Mass HPLC ID #/ expected/retention min. Sequence Target Observedtime 18 5-T*sC*sT*sG*sA*sG*s ICAM 8602.67/31.96 T*sA*sG*sC*sA*sG*sA*s8607.74 G*sG*sA*sG*sC*sT*sC*-3′ 18 5′-T*C*T*G*A*G*T*A*G*C* ICAM8298.66/28.44 A*G*A*G*G*A*G*C*T*C*-3′ 8301.38 5′-GCGTAT*ACG-3′3131.35/21.87 @ 3130.25

HPLC Conditions C-18, Waters 3.9×300 mm, A=50 mM triethyammoniumacetate, pH 7; B=Acetonitrile; 5 to 60% B in 55 min.; flow 1 ML/min., @5 to 18% B in 30 min.; flow 1.5 ML/min., T*=2′-O-DMAOE T, A*=2′-O-DMAOEA, C*=2′-O-DMAOE ^(5me)C, G*=2′-O-DMAOE G.

EXAMPLE 85 O²,2′-anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0, 0.279 mol), diphenylcarbonate (90.0, 0.420 mol)and sodium bicarbonate (2.0 g, 0.024 mol) were added todimethylformamide (300 ML). The mixture was heated to reflux withstirring allowing the resulting carbon dioxide gas to evolve in acontrolled manner. After 1 hour, the slightly darkened solution wasconcentrated under reduced pressure. The resulting syrup was poured intostirred diethyl ether (2.5 L). The product formed a gum. The ether wasdecanted and the residue was dissolved in a minimum amount of methanol(ca 400 Ml). The solution was poured into fresh ether as above (2.5 L)to give a stiff gum. The ether was decanted and the gum was dried in avacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid which wascrushed to a light tan powder (57 g, 85% crude yield). NMR wasconsistent with structure and contamination with phenol and its sodiumsalt (ca 5%). The material was used as is for ring opening. It can bepurified further by column chromatography using a gradient of methanolin ethyl acetate (10-25%) to give a white solid, mp 222-4° C.

EXAMPLE 86 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyl uridine(1a)

O²,2′-Anhydro-5-methyluridine (100.0 g, 0.416 mmol) anddimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved indry pyridine (500 ML) at ambient temperature under an argon atmosphereand with mechanical stirring. Tert-butyldiphenylchlorosilane (125.8 g,119.0 ML, 1.1 eq, 0.458 mmol) was added in one portion and the reactionwas stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethylacetate) indicated a complete reaction. The solution was concentratedunder reduced pressure to a thick oil which was partitioned betweendichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine(1 L). The organic layer was dried over sodium sulfate and concentratedunder reduced pressure to a thick oil. The oil was dissolved in a 1:1mixture of ethyl acetate and ethyl ether (600 ML) and the solution wascooled to −10° C. The resulting crystalline product was collected byfiltration, washed with ethyl ether (3×200 ML) and dried (40° C., 1 mmHg, 24 h) to give 149g (74.8%) of the title compound as a white solid.TLC and NMR were consistent with pure product.

EXAMPLE 87 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (2a)

Borane in THF (1.0 M, 2.0 eq, 622 ML) was added to a 2 L stainlesssteel, unstirred pressure reactor. In the fume hood and with manualstirring, ethylene glycol (350 ML, excess) was added cautiously at firstuntil the evolution of hydrogen gas subsided.5′-O-Tert-butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (R_(f) 0.67 for desired product and Rf 0.82for ara-T side product, ethyl acetate) indicated about 70% conversion tothe product. In order to avoid additional side product formation, thereaction was stopped, concentrated under reduced pressure (10 to 1 mmHg) in a warm water bath (40-100° C.) with the more extreme conditionsused to remove the ethylene glycol. [Alternatively, once the low boilingsolvent is gone, the remaining solution can be partitioned between ethylacetate and water with the product in the organic phase.] The residuewas purified by column chromatography (2 kg silica gel, ethylacetate:hexanes gradient from 1:1 to 4:1). The appropriate fractionswere combined, concentrated and dried to give 84 g (50%) of the titlecompound as a white crisp foam. Also collected from the column wascontaminated starting material (17.4 g) and pure reusable startingmaterial (20 g). The yield, based on starting material less purerecovered starting material, was 58%. TLC and NMR were consistent withthe title compound at a purity of 99%.

¹H NMR (DMSO-d₆) d 1.05 (s, 9H), 1.45 (s, 3 H), 3.5-4.1 (m, 8 H), 4.25(m, 1 H), 4.80 (t, 1 H), 5.18 (d, 2H), 5.95 (d, 1 H), 7.35-7.75 (m, 11H), 11.42 (s, 1 H).

EXAMPLE 882′-O-[2-(phthalimidoxy)ethyl]-5′-tert-butyldiphenylsilyl-5-methyluridine (3a)

Nucleoside 2a (20 g, 36.98 mmol) was mixed with triphenylphosphine(11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). Itwas then dried over P₂O₅ in vacuo for two days at 40° C. The reactionmixture was flushed with argon and dry THF (369.8 ML) was added to givea clear solution. Diethyl azodicarboxylate (6.98 ML, 44.36 mmol) wasadded dropwise to the reaction mixture. The rate of addition wasmaintained such that the resulting deep red coloration is justdischarged before adding the next drop. After the addition was complete,the reaction was stirred for 4 hrs. The TLC showed the completion of thereaction (ethyl acetate:hexane, 60:40). The solvent was evaporated invacuo and the resulting residue was purified by flash columnchromatography using ethyl acetate:hexane (60:40) as the eluent to give21.81 g (86%) of the title compound as a white foam. TLC R_(f) 0.56(ethyl acetate:hexane, 60:40). MS (FAB⁻) m/z 684 (M−H⁺)

EXAMPLE 895′-O-tert-butyldiphenylsilyl-2′-O-[2-(formaldoximinooxy)ethyl]-5-methyluridine (4a)

Methylhydrazine (300 ML, 4.64 mmol) was added dropwise at from −10° C.to 0C to Compound 3a (3.1 g, 4.5 mmol) dissolved in dry CH₂Cl₂ (4.5 ML).After 1 hour the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to give2′-O-(aminooxyethyl) thymidine, which was dissolved in MeOH (67.5 ML).Formaldehyde (20% aqueous solution, w/w, 1.1 eg.) was added and themixture was stirred at room temperature for 1 h. The solvent was removedin vacuo and the residue purified by column chromatography to give 1.95g (78%) of the title compound as a white foam.

R_(f) 0.32 (5% MeOH in CH₂Cl₂). ¹H NMR (200 MHZ, DMSO-d₆) δ1.03 (s, 9H),1.45 (s, 3H) 3.66-4.03 (m, 9H), 5.20 (d, 1H, J=5.92 Hz), 5.91 (d, 1H,J=5.42 Hz), 6.54 (d, 1H, J=7.7 Hz), 6.99 (d, 1H, 7.64 Hz), 7.39-7.48 (m,6H), 7.61-7.67 (m, 4H), 11.39 (s, 1H); ¹³C NMR (50 MHZ, CDCl₃) 11.84,19.41, 62.96, 68.57, 70.02, 72.61, 82.67, 84.33, 87.14, 111.12, 127.90,129.98, 132.37, 133.10, 134.93, 135.18, 135.44, 137.96, 150.50, 164.02;MS (Electrospray) m/z 566 (M−H).

EXAMPLE 905′-O-tert-Butyldiphenylsilyl-2′-O-[2-(N-methyl)aminooxyethyl]-5-methyluridine (5a)

Compound 4a (2.3 g, 4.17 mmol) was dissolved in 1Mpyridinium-p-toluenesulfonate in MeOH (41.7 ML). The reaction mixturewas cooled to 10° C. on an ice bath and NaBH₃CN (0.52 g, 8.35) was addedwith continued cooling and stirring for 15 minutes. The mixture wasallowed to warm to room temperature and stirred for 4 hours. Theprogress of the reaction was complete as indicated by TLC (5% MeOH inCH₂Cl₂). The mixture was concentrated to a syrup and diluted with ethylacetate (50 ML) and washed with water (30 ML), 5% aqueous NaHCO₃ (30 ML)and brine (30 ML). The ethyl acetate layer was dried over anhydrousNa₂SO₄ and evaporated to dryness to give 2.32 g of the title compound asa foam. The foam was used for the next step without furtherpurification. R_(f) (0.34, 5% MeOH in CH₂Cl₂).

EXAMPLE 915′-O-tert-Butyldiphenylsilyl-2′-O-{2-[(N-methyl)-N-(2-phthalimido)ethyl]aminooxyethyl}-5-methyl uridine (6a)

Compound 5a (2 g, 3.44 mmol) was dissolved in 1M pyridinium p-toulenesulfonate in MeOH (34 ML). α-Phthalimidoacetaldehyde (0.72 g, 3.78 mmol)was added and the mixture was stirred at ambient temperature for 10minutes. The reaction mixture was cooled to 10° C. in an ice bath andNaBH₃CN (0.43 g, 0.89 mmol) was added with stirring at 10 ° C. for 15minutes. The reaction mixture was allowed to warm to room temperature,stirred for 4 hours, concentrated to an oil and diluted with ethylacetate (50 ML). The ethyl acetate layer was washed with water (40 ML),5% NaHCO₃ (40 ML) and brine (25 ML). The organic phase was dried overanhydrous Na₂SO₄ and evaporated to dryness. The residue was purified byflash column chromatography and eluted with ethyl acetate:hexane 60:40to give 1.54 g (60%) of the title compound.

R_(f)=0.68 (Ethyl acetate). ¹H NMR (200 MHZ, DMSO-d₆) δ1.04 (s, 9H),1.41 (s, 3H), 2.46 (s, 3H), 2.79 (t, 2H, J=6.34 Hz), 3.69-4.08 (m, 10H),4.27 (m, 1H), 5.22 (d, 1H, J=5.7 MHZ), 5.95 (d, 1H, J=5.86 Hz), 7.39-7.7(m, 11H), 7.84 (s, 4H), 11.38 (s, 1H); HRMS (MALDI) Calcd forC₃₉H₄₆O₉N₄SiNa⁺765.2932, Found: 765.2922.

EXAMPLE 922′-O-{2-[(N-methyl)-N-(2-phthalimido)ethyl]aminooxyethyl}-5-methyluridine (7a)

A solution of triethylamine trihydrogen fluoride (2.64 ML, 16.2 mmol)and triethylamine (1.13 ML, 8.1 mol) in THF was added to compound 6a(1.2 g, 1.62 mmol) with stirring at room temperature for 18 hours. TLCindicated that the reaction was completed at this time (10% MeOH inCH₂Cl₂) The solvent was removed in vacuo, the residue dissolved in ethylacetate (30 ML), the organic layer washed with water (30 ML), brine (30ML) and dried over anhydrous Na₂SO₄. The organic phase was evaporatedand the residue purified by flash chromatography using 5% MeOH in CH₂Cl₂as eluent to give 0.42 g (52%) of the title compound as a solid.

(R_(f)=0.34, 10% MeOH in CH₂Cl₂) ¹H NMR (200 MHZ, DMSO-d₆) δ1.70 (s,3H), 2.46 (s, 3H), 2.78 (t, 2H, J=6.35 Hz), 3.54-3.74 (m, 8H), 3.8 (d,1H, J=3.52 Hz), 3.97 (t, 1H, J=5.26 Hz), 4.10 (q, 1H, J=4.98 Hz), 5.05(d, 1H, J=5.58 Hz), 5.12 (t, 1H, J=5.14 Hz), 5.86 (d, 1H, J=5.64 Hz),7.75 (s, 1H), 7.84 (s, 4H), 11.29 (s, 1H); ¹³C (50 MHZ, CDCl₃) 12.37,35.55, 45.62, 58.05, 61.58, 69.06, 69.97, 70.98, 81.39, 85.2, 90.7,110.67, 123.17, 132.01, 133.94, 138.05, 150.49, 164.24, 168.47; HRMS(FAB) Calcd for C₂₃H₂₉O₉N₄ ^(Å) 505.1927; Found: 505.1927.

EXAMPLE 935′-O-DMT-2′-O-{2-[N-(methyl)-N-(2-phthalimido)ethyl]aminooxyethyl}-5-methyluridine (8a)

Compound 7a (0.4 g, 0.79 mmol), dried over P₂O₅ at 40° C. in vacuoovernight, was mixed with DMAP (0.019 g, 0.16 mmol) and co-evaporatedwith pyridine (3 ML). The residue was dissolved in anhydrous pyridine(1.9 ML) and DMTCl (0.29 g, 0.87 mmol) was added. The reaction mixturewas stirred at room temperature under inert atmosphere for 8 hours withmonitoring by TLC (5% MeOH in CH₂Cl₂). Additional DMTCl (0.15 mg) wasadded with stirring continued until disappearance of the startingmaterial. Pyridine was removed in vacuo and the residue was purified byflash column chromatography using ethyl acetate:hexane 60:40 as theeluent to give 0.47 g (73%) of the title compound.

(R_(f)=0.35, 5% MeOH in CH₂Cl₂). ¹H NMR (200 MHZ, DMSO-d₆) δ1.36 (s,3H), 2.48 (s, 3H), 2.79 (t, 2H, J=6.34 Hz), 3.21 (m, 2H), 3.73 (brs,12H), 3.97 (m, 1H), 4.07 (m 1H), 4.22 (m, 1H), 5.16 (d, 1H, J=6.12 Hz),5.87 (d, 1H, J=4.94 Hz), 6.89 (d, 3H, J=4H), 7.34-7.43 (m, 9H), 7.48(s,1H), 7.83 (s, 4H), 11.36 (s, 1H); ¹³C (50 MHZ, CDCl₃), 11.59, 35.36,45.5, 54.83, 57.94, 61.96, 68.85, 69.86, 82.6, 83.15, 86.47, 87.34,110.53, 112.99, 122.56, 125.80, 127.72, 127.96, 129.8, 131.82, 133.63,135.27, 135.83, 144.18, 150.44, 158.33, 164.28, 168.21; HRMS (FAB) Calcdfor C₄₄H₄₆O₁₁N₄Na^(Å) 829.3061, Found 829.3066.

EXAMPLE 945′-O-DMT-2′-O-{2-[N-(methyl)-N-(2-phthalimido)ethyl]aminooxyethyl}-5-methyl-uridine-3′-O-[(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite(9a)

N,N-Diisopropylamine tetrazolide (0.055 g, 0.32 mmol, dried over P₂O₅ invacuo at 40° C. overnight) was added to Compound 8a (0.26 g, 0.32 mmol,co-evaporated with toulene) followed by anhydrous acetonitrile (1.6 ML)with stirring at room temperature for 18 hours under an inertatmosphere. Analysis by TLC (ethylacetate:hexane 60:40) showed thereaction was completed at this time. The solvent was remove in vacuo andthe residue was purified by flash column chromatography using ethylacetate containing 0.5% of pyridine as the eluent to give 0.28 g (85%)of the title compound.

(R_(f)=0.28, ethylacetate:hexane, 60:40). ³¹P NMR (80 MHZ, CDCl₃)δ150.82, 150.61; MS (FAB) m/z 1029 [M+Na]^(Å).

EXAMPLE 955′-O-DMT-2′-O-{2-[N-(methyl)-N-(2-phthalimido)ethyl]aminooxyethyl}-3′-O-[(2-succinyl-5-methyluridine (10a)

Compound 8a (0.16 g, 0.2 mmol) was mixed with DMAP (0.013 g, 0.10 mmol)and succinic anhydride (0.03 g, 0.3 mmol) and dried over P₂O₅ in vacuoat 40° C. overnight. CH₂Cl₂ (0.5 ML) and triethylamine (0.06 ML, 0.4mmol) was added with stirring at room temperature for 4 hours under aninert atmosphere. The mixture was diluted with CH₂Cl₂ (30 ML) and washedwith 10% aqueous citric acid (30 ML) and water (2×15 ML). The organiclayer was dried over anhydrous Na₂SO₄ and concentrated to give 0.162 g(90%) of the title compound as a foam.

(R_(f)=0.43, 10% MeOH in CH₂Cl₂). ¹H NMR (200 MHZ, DMSO-d₆) δ1.4 (s,3H), 2.42 (s, 3H), 2.56 (m, 4H, overlap with DMSO peak), 2.75 (t, 2H,J=6.29 Hz), 3.24 (m, 2H, overlapping with H₂O peak), 3.53-3.8 (m, 6H),3.72 (s, 6H), 4.13 (brs, 1H), 4.37 (t, 1H, J=5.86 Hz), 5.29 (t, 1H,J=4.4 Hz), 5.87 (d, 1H, J=6.36 Hz), 6.89 (d, 4H, J=8.72 Hz), 7.21-7.39(m, 9H), 7.49 (s, 1H), 7.82 (s, 4H), 11.42 (s, 1H), 12.24 (brs, 1H); MS(FAB) m/z 929 [M+Na]^(Å).

EXAMPLE 965′-O-DMT-2′-O-{2-[N-(methyl)-N-(2-phthalimido)ethyl]aminooxyethyl]-5-methyl-uridine-3′-O-succinylCPG (11a)

Compound 10a (0.15 g, 0.17 mmol) and DMAP (0.021 g, 0.17 mmol) wasdissolved in anhydrous acetonitrile. To protect the reaction mixturefrom moisture 2,2′-dithiobis(5-nitropyridine) (0.068 g, 0.19 mg) wasadded. The solution was stirred for 5 minutes at room temperature. Tothis solution triphenyl phosphine (0.045 g, 0.17 mmol) in anhydrousacetonitrile (1.12 ML) was added. The solution was stirred for 10minutes at ambient temperature. Activated CPG (Controlled Pore Glass,1.12 g, 115.2 mmol/g, particle size 120/200, mean pore diameter 520 Å)was added and allowed to shake on a shaker for 2 hours. An aliquot waswithdrawn and loading capacity was determined by following standardprocedure (61.52 mmol/g). The functionalized CPG (11a) was filtered andwashed throughly with CH₃CN, CH₂Cl₂ and Et₂O. It was then dried in vacuoover night. Any unreacted sites on the CPG was capped by using cappingreagents [CapA (2 ML), acetic anhydride/leuticle/THF; Cap B (2 ML),1-methylimidazole/THF, PerSeptive Biosystems Inc.] and allowed to shakeon a shaker for 2 h. The functionalized CPG was filtered, washedthoroughly with CH₃CN, CH₂Cl₂ and Et₂O. It was then dried and the finalloading capacity was determined (60.74 mmol/g).

EXAMPLE 975′-O-DMT-2′-O-{2-[(N,N-bis-2-phthalimidoethyl)aminooxy]ethyl}-5-methyluridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl] phosphoramidite (12a)

Compound 4a is treated with methylhydrazine to give the aminooxycompound followed by treatment with phtalimidoacetaldehyde to give thecorresponding oxime. The oxime is reduced under acid catalyzed reductiveamination conditions to give the 2-phtalimidoethyl derivative which ontreatment with another equivalent of phtalimidoacetaldehyde underreductive amination conditions will give thebis(2-phthalimidoethyl)aminooxyethyl derivative. Thebis(2-phthalimidoethyl)aminooxyethyl derivative is desilylated,tritylated at 5′-position and then 3′-phosphitylated to give the titlecompound.

EXAMPLE 985′-O-DMT-2′-O-{2-[(N,N-bis-2-phthalimidoethyl)aminooxy]ethyl}-5-methyluridine-3′-O-succinyl CPG (13a)

Compound 14a is synthesized according to the procedure described forcompounds 11a and 12a starting from5′-O-DMT-2′-O-{2-[N,N-bis-(2-phthalimido)ethylaminooxy]ethyl}-5-methyluridine.

EXAMPLE 99 Synthesis of oligonucleotides containing2′-O-{2-[N-(2-amino)ethyl-N-(methyl)]aminooxyethyl} modification

Phosphoramidite 9a was dissolved in anhydrous acetonitrile (0.1 Msolution) and loaded on to a Expedite Nucleic Acid Synthesis system(Millipore 8909) for use in oligonucleotide synthesis. The couplingefficiencies were determined to be greater than 98%. For the coupling ofthe modified phosphoramidite 9a coupling time was extended to 10 minutesand this step was carried out twice. All other steps in the protocolsupplied by Millipore were used without modification. After completionof the synthesis CPG was suspended in aqueous ammonia solution (30 wt %)containing 10% methyl amine (40 wt % solution) and heated at 55° C. for6 h. The resulting oligonucleotides were purified by HPLC (Waters, C-4,7.8×300 mm, A=50 mM triethylammonium acetate, pH=7, B acetonitrile, 5 to60% B in 55 Min, Flow 2.5 ML/min., λ=260 nm). Detritylation with aqueous80% acetic acid and evaporation followed by desalting by HPLC on WatersC-4 column gave 2′-modified oligonucleotides (Table I). Oligonucleotideswere analyzed by HPLC, CGE and mass spectrometry.

TABLE I Oligonucleotides containing2′-O-{2-[N-(2-amino)ethyl-N-(methyl)] aminooxyethyl} modification SEQ IDHPLC No./ Mass Mass Retention ISIS # Sequence Calcd Found Time(min.^(a)) 6/ 5′ CTC GTA CT*T* 5919.21 5919.79 23.79^(a) 30443 T*T*C CGGTCC 3′ 14/ 5′ TTT TTT TTT TTT 6246.45 6243.04 25.56^(b) 26267 TTT T*T*T*T* 3′ T* = 2′-O-{2-[N-(2-amino)ethyl-(N-methyl)aminooxy]ethyl} ^(5Me)U

7.8×300 mm, solvent A=50 mm TEAAc, pH 7; Solvent B=CH₃CN; gradient 5-60%B in 50 min; flow rate 2.5 ML/min, 1=260 nm, 3.9×300 mm, solvent A=50 mmTEAAC, pH 7; Solvent B=CH₃CN; gradient 5-40% B in 55 min; flow rate 1.5ML/min, 1=260 nm.

TABLE II Tm values of 2′-O-{2-[N-(2-amino)ethyl-(N-methyl)aminooxy]ethyl} modifications SEQ ID Tm ° C. No./ SequenceTarget ΔTm ΔTm/mod ISIS # 5′-3′ RNA ° C. ° C. 6/ CTC GTA CTT TTC CGG TCC61.8 2896 6/ CTC GTA CT*T* T*T*C CGG 63.60 1.8 0.38 32350 TCC T* =2′-O-{2-[N-(2-amino)ethyl-N-(methyl)aminooxy]ethyl} ^(5Me)U

EXAMPLE 1005′-O-DMT-2′-O-{2-[N-2-(N,N-dimethylamino)ethyl-N-(methyl)aminooxy]ethyl}-5-methyluridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite (14a)

Compound 14a is synthesized from compound 3a. The phthalimido compound3a is deprotected with methylhydrazine to form the aminooxy compound.The reactive aminooxy compound is treated withα-(N,N-dimethylamino)acetaldehyde diethyl acetal to give thecorresponding oxime. The oxime is reduced under acid catalyzed reductiveamination conditions to give the2-{[2-N,N-(dimethyl)amino]ethylaminooxy}ethyl derivative which ontreatment with formaldehyde under reductive amination condition givesthe 2-{[N-2-(N,N-dimethyl)amino]ethyl-N-(methyl)aminooxy}ethylderivative. Desilylation, tritylation and phosphitylation as illustratedin previous examples gives the title phosphoramidite.

Compound 101

5′-O-DMT-2′-O-{2-[N-2-(N,N-dimethylamino)ethyl-N-(methyl)-aminooxy]ethyl}-5-methyluridine-3′-O-succinyl CPG (15a)

Compound 15a is synthesized according to the procedure described forcompounds 11a and 12a starting from5′-O-DMT-2′-O-{2-[N-(2-N,N-dimethylamino)ethyl-N-(methyl)aminooxy]ethyl}-5-methyluridine.

Procedure 1

Nuclease Resistance

A. Evaluation of the resistance of modified oligonucleotides to serumand cytoplasmic nucleases.

Oligonucleotides including the modified oligonucleotides of theinvention can be assessed for their resistance to serum nucleases byincubation of the oligonucleotides in media containing variousconcentrations of fetal calf serum or adult human serum. Labeledoligonucleotides are incubated for various times, treated with proteaseK and then analyzed by gel electrophoresis on 20% polyacrylamide-ureadenaturing gels and subsequent autoradiography. Autoradiograms arequantitated by laser densitometry. Based upon the location of themodifications and the known length of the oligonucleotide it is possibleto determine the effect on nuclease degradation by the particularmodification. For the cytoplasmic nucleases, a HL60 cell line is used. Apost-mitochondrial supernatant is prepared by differentialcentrifugation and the labeled oligonucleotides are incubated in thissupernatant for various times. Following the incubation,oligonucleotides are assessed for degradation as outlined above forserum nucleolytic degradation. Autoradiography results are quantitatedfor comparison of the unmodified and modified oligonucleotides. As acontrol, unsubstituted phosphodiester oligonucleotide have been found tobe 50% degraded within 1 hour, and 100% degraded within 20 hours.

B. Evaluation of the resistance of modified oligonucleotides to specificendo- and exonucleases.

Evaluation of the resistance of natural and modified oligonucleotides tospecific nucleases (i.e., endonucleases, 3′,5′-exo-, and5′,3′-exonucleases) is done to determine the exact effect of themodifications on degradation. Modified oligonucleotides are incubated indefined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea is added andanalysis on 20% polyacrylamide gels containing urea is done. Gelproducts were visualized by staining using Stains All (Sigma ChemicalCo.). Laser densitometry is used to quantitate the extend ofdegradation. The effects of the modifications are determined forspecific nucleases and compared with the results obtained from the serumand cytoplasmic systems.

Nuclease resistance of oligonucleotides containing novel2′-modifications

SEQ. ID NO: 14 Series I 5′TTT TTT TTT TTT TTT*T*T*T* T 3′ SEQ ID whereT* = 5 methyl, 2′- 2′ AOE NO 14 aminooxyethoxy SEQ ID where T* = 5methyl, 2′- 2′ DMAOE NO 14 dimethylaminooxyethoxy

Along with T19 diester and thioate controls, the gel purified oligoswere 5′ end labeled with ³²P, and run through the standard nucleaseassay protocol. PAGE/Phosphorimaging generated images that werequantified for % Intact and % (Intact+(N−1)). The percentages wereplotted to generate half-lives, which are listed in a table below.Included is the half life of the 2′-O-methoxyethyl (MOE) analog in thetable. This result showed that 2′-dimethylaminooxyethyl (DMAOE) is ahighly nuclease resistant modification (FIGS. 14 and 15).

2′-Modification AOE DMAOE MOE T1/2 of N  18 60 100 (min) T1/2 of N + (N− 200 85% remaining at 300 1) (min) 24 hr.

Initial assays of the nuclease resistance of oligonucleotides cappedwith 2′-DMAOE modifications showed better resistance than modification2′-O-methoxyethyl in an inter-assay comparison (FIG. 13). These studiesare intra-assay comparisons among several modifications in two motifs.The first motif is a full phosphodiester backbone, with a cap of 4modified nucleotides beginning at the 3′-most nucleotide. The secondmotif is similar, but contains a single phosphorothioate at the 3′-mostinter nucleotide linkage.

SEQ. ID NO: 14 Series II 5′ TTT TTT TTT TTT TTT T*T*T* T* 3′ SEQ IDwhere T* = 2′-O- NO 14 dimethylaminooxyethyl SEQ ID where T* =2′-O-methoxyethyl NO 14 SEQ ID where T* = 2′-O-propyl NO 14 SEQ. ID NO:14 Series III 5′ TTT TTT TTT TTT TTT TTT*T 3′ SEQ ID where T* = 5methyl, 2′- NO 14 dimethylaminooxyethyl SEQ ID where T* = 5 methyl,2′-O- NO 14 methoxyethyl

Along with a T19 phosphorothioate control, the oligos were gel purifiedand run through the standard nuclease protocol. From these assays SEQ IDNO: 14 where T*=2′-O-dimethylaminooxyethyl proved to be the next mostresistant oligonucleotide. SEQ ID NO: 14 where T*=2′-O-methoxyethyl wasdegraded more readily and SEQ ID NO: 14 where T*=2′-O-propyl is degradedrather quickly. The gel shows some reaction products at the bottom ofthe gel, but little n-2 and n-3 of the resistant oligonucleotides. Theseproducts appear to be the result of endonucleolytic cleavage by SVPD.This type of activity is always present at a basal rate, but is notusually seen due to the overwhelming predominance of 3′ exonucleaseactivity on most oligonucleotides. However, these oligonucleotides areso extraordinarily resistant to 3′ exonucleases that the endonucleaseactivity is responsible for a majority of the cleavage events on thefull-length oligo. 2′-deoxy phosphodiester products of the endonucleasereactions are then rapidly cleaved to monomers. Two sets of quantitationare done for these reactions. One counts only 3′-exonuclease products,and the other counts products for all reactions. In either case, thehalf-life of SEQ ID NO: 14 where T*=2′-O-dimethylaminooxyethyl waslonger than 24 hours. For SEQ ID NO: 14 where T*=2′-O-methoxyethyl thehalf life upon treatment with exonuclease is over 24 hours while theother type of quantitation gives a half-life of about 100 min. Theoligonucleotides of the motif containing a single phosphorothioatelinkage are substrates for the endonuclease activity described above,but no products of 3′ exonuclease activity are detected in the timecourse of this assay.

TABLE 2 Oligonucleotides synthesized with 2′-dimethylaminooxyethylthymidine (T-2′-DMAOE) SEQ ID Mass NO: Sequence Exp. Obs.  5 5′-CTCGTACCT*TTCCGGTCC-3′ 5784.20 5784.09 15 5′-T*CCAGGT*GT*CCGCAT*C-3′5548.74 5549.05  3 5′-GCGT*T*T*T*T*T*T*T*T*T*GCG-3′ 6208.74 6210.52 145′-TTTTTTTTTTTTTTT*T*T*T*T-3′ 6433.45 6433.79 N/A 5′-T*T*T*T*-3′ 1869.961869.5 14 5′-TTTTTTTTTTTTTTTT*T*T*_(S)T*-3′ 6449.45 6449.15 14TTTTTTTTTTTTTTTT*T*T*T*-3′ 6433.51 6433.19 N/A 5′-T*T*-3′ 648.49 648.4

TABLE 3 Oligonucleotides synthesized with 2′-dimethylaminooxyethyladenosine (A-2′-DMAOE) SEQ ID Mass NO: Sequence Exp. Obs. 1 7 5′-CTCGTACCA*TTCCGGTCC-3′ 5490.21 5490.86 2 8 5′-GGA*CCGGA*A*GGTA*CGA*G-3′5824.96 5826.61 3 16  5′-A*CCGA*GGA*GGA*TCA*TGTCGTA*CGC-3′ 6947.96947.28

TABLE 4 Oligonucleotides synthesized with 2′-O-methyleneiminooxyethyladenosine SEQ ID Mass NO: Sequence Exp. Obs. 7 5′-CTCGTACCA*TTCCGGTCC-3′5470.20 5472.50 17  5′-A*CCGA*GGA*TCA*TGTCGTA*CGC-3′ 6866.42 6865.88 85′-GGA*CCGGA*A*GGTA*CGA*G-3′ 5743.12 5743.82

TABLE 5 Oligonucleotides synthesized with 2′-O-methyleneiminooxyethylthymidine SEQ ID Mass NO: Sequence Exp. Obs. 1  55′-CTCGTACCT*TTCCGGTCC-3′ 5466.21 5462.25 2 155′-T*CCAGGT*GT*CCGCAT*C-3′ 5179.44 5178.96 3 145′-TTTTTTTTTTTTTTT*T*T*T*T-3′ 6369.45 6367.79

TABLE 6 Tm advantage of 2′-DMAOE modification over 2′-deoxyphosphodiesters and phosphorothioates ΔTm/mod against ΔTm/mod RNAagainst compared to RNA compared unmodified SEQ. to deoxy- ID unmodifiedphosphoro- NO: SEQUENCE Tm DNA thioate 5 5′-CTCGTAC-CT*T- 65.44 0.241.04 TCCGGTCC-3′ 15  5′-T*CCAGGT*GT*C- 67.90 1.12 2.20 CGCAT*C-3′ 35′-GCGT*T*T*T*T*T* 62.90 1.46 2.36 T*T*T*T*GCG-3′

ΔTm is based on reported literature values for DNA and phosphorothioateoligonucleotides.

Procedure 2

Ras-Luciferase Reporter Gene Assembly

The ras-luciferase reporter genes described in this study are assembledusing PCR technology. Oligonucleotide primers are synthesized for use asprimers for PCR cloning of the 5′-regions of exon 1 of both the mutant(codon 12) and non-mutant (wild-type) human H-ras genes. H-ras genetemplates are purchased from the American Type Culture Collection (ATCCnumbers 41000 and 41001) in Bethesda, Md. The oligonucleotide PCRprimers5′-ACA-TTA-TGC-TAG-CTT-TTT-GAG-TAA-ACT-TGT-GGG-GCA-GGA-GAC-CCT-GT-3′(sense) (SEQ ID NO:10), and 5′-GAG-ATC-TGA-AGC-TTC-TGG-ATG-GTC-AGC-GC-3′(antisense) (SEQ ID NO:11), are used in standard PCR reactions usingmutant and non-mutant H-ras genes as templates. These primers areexpected to produce a DNA product of 145 base pairs corresponding tosequences −53 to +65 (relative to the translational initiation site) ofnormal and mutant H-ras, flanked by NheI and HindIII restrictionendonuclease sites. The PCR product is gel purified, precipitated,washed and resuspended in water using standard procedures.

PCR primers for the cloning of the P. pyralis (firefly) luciferase genewere designed such that the PCR product would code for the full-lengthluciferase protein with the exception of the amino-terminal methionineresidue, which would be replaced with two amino acids, an amino-terminallysine residue followed by a leucine residue. The oligonucleotide PCRprimers used for the cloning of the luciferase gene are5′-GAG-ATC-TGA-AGC-TTG-AAG-ACG-CCA-AAA-ACA-TAA-AG-3′ (sense) (SEQ IDNO:12), and 5′-ACG-CAT-CTG-GCG-CGC-CGA-TAC-CGT-CGA-CCT-CGA-3′(antisense) (SEQ ID NO:13), are used in standard PCR reactions using acommercially available plasmid (pT3/T7-Luc) (Clontech), containing theluciferase reporter gene, as a template. These primers are expected toyield a product of approximately 1.9 kb corresponding to the luciferasegene, flanked by HindIII and BssHII restriction endonuclease sites. Thisfragment is gel purified, precipitated, washed and resuspended in waterusing standard procedures.

To complete the assembly of the ras-luciferase fusion reporter gene, theras and luciferase PCR products are digested with the appropriaterestriction endonucleases and cloned by three-part ligation into anexpression vector containing the steroid-inducible mouse mammary tumorvirus promotor MMTV using the restriction endonucleases NheI, HindIIIand BssHII. The resulting clone results in the insertion of H-ras 5′sequences (−53 to +65) fused in frame with the firefly luciferase gene.The resulting expression vector encodes a ras-luciferase fusion productwhich is expressed under control of the steroid-inducible MMTV promoter.

Procedure 3

Transfection of Cells with Plasmid DNA

Transfections are performed as described by Greenberg in CurrentProtocols in Molecular Biology, Ausubel et al., Eds., John Wiley andSons, New York, with the following modifications: HeLa cells are platedon 60 mm dishes at 5×10⁵ cells/dish. A total of 10 μg of DNA is added toeach dish, of which 9 μg is ras-luciferase reporter plasmid and 1 μg isa vector expressing the rat glucocorticoid receptor under control of theconstitutive Rous sarcoma virus (RSV) promoter. Calcium phosphate-DNAcoprecipitates are removed after 16-20 hours by washing withTris-buffered saline (50 Mm Tris-Cl (pH 7.5), 150 mM NaCl) containing 3mM EGTA. Fresh medium supplemented with 10% fetal bovine serum is thenadded to the cells. At this time, cells are pre-treated with antisenseoligonucleotides prior to activation of reporter gene expression bydexamethasone.

Procedure 4

Oligonucleotide Treatment of Cells

Immediately following plasmid transfection, cells are thrice washed withOptiMEM (GIBCO), and prewarmed to 37° C. 2 ML of OptiMEM containing 10μg/ML N-[1-(2,3-diolethyloxy)propyl]-N,N,N,-trimethylammonium chloride(DOTMA) (Bethesda Research Labs, Gaithersburg, Md.) is added to eachdish and oligonucleotides are added directly and incubated for 4 hoursat 37° C. OptiMEM is then removed and replaced with the appropriate cellgrowth medium containing oligonucleotide. At this time, reporter geneexpression is activated by treatment of cells with dexamethasone to afinal concentration of 0.2 μM. Cells are harvested 12-16 hours followingsteroid treatment.

Procedure 5

Luciferase Assays

Luciferase is extracted from cells by lysis with the detergent TritonX-100, as described by Greenberg in Current Protocols in MolecularBiology, Ausubel et al., Eds., John Wiley and Sons, New York. A DynatechML1000 luminometer is used to measure peak luminescence upon addition ofluciferin (Sigma) to 625 μM. For each extract, luciferase assays areperformed multiple times, using differing amounts of extract to ensurethat the data are gathered in the linear range of the assay.

Procedure 6

Antisense Oligonucleotide Inhibition of ras-Luciferase Gene Expression

A series of antisense phosphorothioate oligonucleotide analogs targetedto the codon-12 point mutation of activated H-ras are tested using theras-luciferase reporter gene system described in the foregoing examples.This series comprised a basic sequence and analogs of that basicsequence. The basic sequence is of known activity as reported inInternational Publication Number WO 92/22651 identified above. In boththe basic sequence and its analogs, each of the nucleotide subunitsincorporated phosphorothioate linkages to provide nuclease resistance.Each of the analogs incorporated nucleotide subunits that contained2′-O-substitutions and 2′-deoxy-erythro-pentofuranosyl sugars. In theanalogs, a subsequence of the 2′-deoxy-erythro-pentofuranosylsugar-containing subunits is flanked on both ends by subsequences of2′-O-substituted subunits. The analogs differed from one another withrespect to the length of the subsequence of the2′-deoxy-erythro-pentofuranosyl sugar containing nucleotides. The lengthof these subsequences are varied by 2 nucleotides between 1 and 9 totalnucleotides. The 2′-deoxy-erythro-pentofuranosyl nucleotidesub-sequences are centered at the point mutation of the codon-12 pointmutation of the activated ras.

Procedure 7

Diagnostic Assay for the Detection of mRNA Overexpression

Oligonucleotides are radiolabeled after synthesis by ³²P labeling at the5′ end with polynucleotide kinase. Sambrook et al. (“Molecular Cloning.A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 1989, Volume2, pg. 11.31-11.32). Radiolabeled oligonucleotide is contacted withtissue or cell samples suspected of mRNA overexpression, such as asample from a patient, under conditions in which specific hybridizationcan occur, and the sample is washed to remove unbound oligonucleotide. Asimilar control is maintained wherein the radiolabeled oligonucleotideis contacted with normal cell or tissue sample under conditions thatallow specific hybridization, and the sample is washed to remove unboundoligonucleotide. Radioactivity remaining in the sample indicates boundoligonucleotide and is quantitated using a scintillation counter orother routine means. Comparison of the radioactivity remaining in thesamples from normal and diseased cells indicates overexpression of themRNA of interest.

Radiolabeled oligonucleotides of the invention are also useful inautoradiography. Tissue sections are treated with radiolabeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to standard autoradiography procedures.A control with normal cell or tissue sample is also maintained. Theemulsion, when developed, yields an image of silver grains over theregions overexpressing the mRNA, which is quantitated. The extent ofmRNA overexpression is determined by comparison of the silver grainsobserved with normal and diseased cells.

Analogous assays for fluorescent detection of mRNA expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling, Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorescence microscope is used to detect the fluorescence.Comparison of the fluorescence observed in samples from normal anddiseased cells enables detection of mRNA overexpression.

Procedure 8

Detection of Abnormal mRNA Expression

Tissue or cell samples suspected of expressing abnormal mRNA areincubated with a first ³²P or fluorescein-labeled oligonucleotide whichis targeted to the wild-type (normal) mRNA. An identical sample of cellsor tissues is incubated with a second labeled oligonucleotide which istargeted to the abnormal mRNA, under conditions in which specifichybridization can occur, and the sample is washed to remove unboundoligonucleotide. Label remaining in the sample indicates boundoligonucleotide and can be quantitated using a scintillation counter,fluorimeter, or other routine means. The presence of abnormal mRNA isindicated if binding is observed in the case of the second but not thefirst sample.

Double labeling can also be used with the oligonucleotides and methodsof the invention to specifically detect expression of abnormal mRNA. Asingle tissue sample is incubated with a first ³²P-labeledoligonucleotide which is targeted to wild-type mRNA, and a secondfluorescein-labeled oligonucleotide which is targeted to the abnormalmRNA, under conditions in which specific hybridization can occur. Thesample is washed to remove unbound oligonucleotide and the labels aredetected by scintillation counting and fluorimetry. The presence ofabnormal mRNA is indicated if the sample does not bind the ³²P-labeledoligonucleotide (i.e., is not radioactive) but does retain thefluorescent label (i.e., is fluorescent).

Procedure 9

Binding Affinity of DMAOE Vs. 2′-deoxyphosphorothioate

The binding affinities of oligonucleotides having either 4 or 10 DMAOEmodifications (SEQ ID NO's: 15 and 2) versus each of 3 complementarysequences was determined. The complementary sequences were a) MOEphosphodiesters with each MOE oligonucleotide substituted at the samepositions as the DMAOE oligonucleotides; b) a uniform 2′-deoxyphosphodiester; and c) a uniform 2′-deoxyphosphorothioate. The DMAOEmodified oligonucleotides show nearly 2.5° C. increase in Tm for eachmodification compared to the uniform 2′-deoxy phosphorothioate. Comparedto the unmodified uniform 2′-deoxy phosphodiester the DMAOEoligonucleotides showed about a 1.6° C. increase in Tm. This willtranslate into 2.5° C./modification compared to the P═S uniform2′-deoxyphosphorothioate DNA. More importantly, this increase is evenhigher than the 2′-MOE by 0.4° C./modification, which is surprising inview of the larger size of DMAOE compared to MOE oligonucleotides.

TABLE 7 Binding Affinity Advantage of 2′-DMAOE over 2′-MOE (P = O),2′-deoxyphosphodiester and 2′-deoxyphosphorothioate SEQ ID Tm vs. Tm vs.2′-H Tm vs. 2′-H number of NO: MOE, ° C. (P = O), ° C. (P = S), ° C.mods 15 0.4 1.6 2.4 4  2 0.4 1.7 2.5 10 

Procedure 10

Procedure A

ICAM-1 Expression

Oligonucleotide Treatment of HUVECs. Cells were washed three times withOpti-MEM (Life Technologies, Inc.) prewarmed to 37° C. Oligonucleotideswere premixed with 10 μg/ML Lipofectin (Life Technologies, Inc.) inOpti-MEM, serially diluted to the desired concentrations, and applied towashed cells. Basal and untreated (no oligonucleotide) control cellswere also treated with Lipofectin. Cells were incubated for 4 h at 37°C., at which time the medium was removed and replaced with standardgrowth medium with or without 5 mg/ML TNF-a (R&D Systems). Incubation at37° C. was continued until the indicated times.

Quantitation of ICAM-1 Protein Expression by Fluorescence-activated CellSorter. Cells were removed from plate surface by brief trypsinizationwith 0.25% trypsin in PBS. Trypsin activity was quenched with a solutionof 2% bovine serum albumin and 0.2% sodium azide in PBS (+Mg/Ca). Cellswere pelleted by centrifugation (1000 rpm, Beckman GPR centrifuge),resuspended in PBS, and stained with 3 μL/10⁵ cells of the ICAM-1specific antibody, CD54-PE (Pharmingin). Antibodies were incubated withthe cells for 30 min at 4° C. in the dark, under gentle agitation. Cellswere washed by centrifugation procedures and then resuspended in 0.3 MLof FacsFlow buffer (Becton Dickinson) with 0.5% formaldehyde(Polysciences). Expression of cell surface ICAM-1 was then determined byflow cytometry using a Becton Dickinson FACScan. Percentage of thecontrol ICAM-1 expression was calculated as follows:[(oligonucleotide-treated ICAM-1 value)-(basal ICAM-1value)/(non-treated ICAM-1 value)-(basal ICAM-1 value))]. In one study,2′-O-(2-methoxy)ethyl-modified anti-intercellular adhesion molecule 1(ICAM-1) oligonucleotides were shown to selectively increase the ICAM-1mRNA level and inhibit formation of the ICAM-1 translation initiationcomplex in human umbilical vein endothelial cells (Baker, et al., TheJournal of Biological Chemistry, 1997, 272, 11994-12000).

ICAM-1 expression data reveal that the DMAOE oligomers SEQ ID NO: 21(uniform DMAOE, P═S) and SEQ ID NO: 18 (uniform DMAOE, P═O) areefficacious in HUVEC cells in controlling ICAM-1 expression. Theoligomers are presumably working by a direct binding RNase H independentmechanism. The MOE oligomers having SEQ ID NO: 21 (P═S) and SEQ ID NO:21 (P═O) stand as controls. They have the same sequence composition asSEQ ID NO: 21 and SEQ ID NO: 18.

Both compounds SEQ ID NO: 21 and SEQ ID NO: 18 display dose response ininhibiting ICAM-1 expression between 3 and 100 nM range.

Procedure B

PKC-a mRNA Expression in A549 Cells

This assay was carried out according to a reported procedure (Dean, N.et al., Journal of Biology and Chemistry, 269, 16416-16424, 1994). HumanA549 lung carcinoma cells were obtained from the American Type TissueCollection. These were grown in Dulbecco's modified Eagle's mediumcontaining 1 g of glucose/liter (DMEM) and 10% FCS and routinelypassaged when 90-95% confluent.

Assay for Oligonucleotide Inhibition of PKC-a Protein Synthesis. A549cells were plated in 6-well plates (Falcon Labware, Lincoln Park, N.J.)and 24-48 h later (when 80-90% confluent) treated with 1 μM phorbol12,13-dibutyrate (PDBu) for 18 h. This procedure removes greater than75% of immunoreactive PKC-a protein from the cells (see “Results”).Cells were then washed three times with 3 ML of DMEM (to remove PDBu),and 1 ML of DMEM containing 20 μg/ML DOTMA/DOPE solution(Lipofectin^(R)) (Bethesda Research Laboratories) was added.Oligonucleotide was then added to the required concentration (for ourinitial screen, 1 μM) from a 10 μM stock solution, and the two solutionswere mixed by swirling of the dish. The cells were incubated at 37° C.for 4 h, washed once with DMEM +10% FCS to remove the DOTMA/DOPEsolution, and then an additional 3 ML of DMEM+10% FCS was added and thecells were allowed to recover for another 10 h. More prolongedincubation times with DOTMA/DOPE solution resulted in increased cellulartoxicity. At this time, cells were washed once in PBS and then extractedin 200 μL of lysis buffer consisting of 20 mM Tris (pH 7.4), 1% TritonX100, 5 mM EGTA, 2 mM dithiothreitol, 50 mM sodium fluoride, 10 mMsodium phosphate, leupeptin (2 μg/ML), and aprotinin (1 μg/ML) (at 4°C.). PKC-a protein levels were determined by immunoblotting with a PKC-aspecific monoclonal antibody. Results: DMAOE oligonucleotide gapmers SEQID NO: 19 (P═S/P═S/P═S gapmer) and SEQ ID NO: 19 (P═O/P═S/P═O gapmers)inhibit PKC-a mRNA expression in A549 cells in a dose dependent mannerbetween 50-400 nM range. The uniform P═S gapmer is more efficacious thanthe mixed backbone gapmer. In this experiment the corresponding MOEoligomers were used as the control compounds. The DMAOE oligomers andthe MOE oligomers exhibit similar activity in reducing PKC-α mRNAlevels.

It is intended that each of the patents, applications, printedpublications, and other published documents mentioned or referred to inthis specification be herein incorporated by reference in theirentirety.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

21 1 10 DNA Artificial Sequence Description of Artificial Sequenceantisense sequence 1 tttttttttt 10 2 25 DNA Artificial SequenceDescription of Artificial Sequence antisense sequence 2 tgcatcccccaggccaccat ttttt 25 3 16 DNA Artificial Sequence Description ofArtificial Sequence antisense sequence 3 gcgttttttt tttgcg 16 4 19 DNAArtificial Sequence Description of Artificial Sequence antisensesequence 4 cgcaaaaaaa aaaaaacgc 19 5 18 DNA Artificial SequenceDescription of Artificial Sequence antisense sequence 5 ctcgtacctttccggtcc 18 6 18 DNA Artificial Sequence Description of ArtificialSequence antisense sequence 6 ctcgtacttt tccggtcc 18 7 18 DNA ArtificialSequence Description of Artificial Sequence antisense sequence 7ctcgtaccat tccggtcc 18 8 17 DNA Artificial Sequence Description ofArtificial Sequence antisense sequence 8 ggaccggaag gtacgag 17 9 21 DNAArtificial Sequence Description of Artificial Sequence antisensesequence 9 accgaggatc atgtcgtacg c 21 10 29 DNA Artificial SequenceDescription of Artificial Sequence antisense sequence 10 acattatgctagctttttga gtaaacttg 29 11 29 DNA Artificial Sequence Description ofArtificial Sequence antisense sequence 11 gagatctgaa gcttctggatggtcagcgc 29 12 35 DNA Artificial Sequence Description of ArtificialSequence antisense sequence 12 gagatctgaa gcttgaagac gccaaaaaca taaag 3513 33 DNA Artificial Sequence Description of Artificial Sequenceantisense sequence 13 acgcatctgg cgcgccgata ccgtcgacct cga 33 14 18 DNAArtificial Sequence Description of Artificial Sequence antisensesequence 14 tttttttttt tttttttt 18 15 16 DNA Artificial SequenceDescription of Artificial Sequence antisense sequence 15 tccaggtgtccgcatc 16 16 24 DNA Artificial Sequence Description of ArtificialSequence antisense sequence 16 accgaggagg atcatgtcgt acgc 24 17 21 DNAArtificial Sequence Description of Artificial Sequence antisensesequence 17 accgaggatc atgtcgtacg c 21 18 20 DNA Artificial SequenceDescription of Artificial Sequence antisense sequence 18 tctgagtagcagaggagctc 20 19 19 DNA Artificial Sequence Description of ArtificialSequence antisense sequence 19 ttctcgctgg tgagtttca 19 20 20 DNAArtificial Sequence Description of Artificial Sequence antisensesequence 20 ttctcgcccg ctcctcctcc 20 21 19 DNA Artificial SequenceDescription of Artificial Sequence antisense sequence 21 ttgagtagcagaggagctc 19

What is claimed is:
 1. A compound of the structure:

wherein: T₄ is Bx or Bx—L where Bx is a heterocyclic base moiety; one ofT₁, T₂ and T₃ is L, hydrogen, hydroxyl, a protected hydroxyl or a sugarsubstituent group; another one of T₁, T₂ and T₃ is L, hydroxyl, aprotected hydroxyl, a connection to a solid support or an activatedphosphorus-containing substituent group; the remaining one of T₁, T₂ andT₃ is L, hydrogen, hydroxyl or a sugar substituent group provided thatat least one of T₁, T₂, or T₃ is L or Bx—L; said group L having one ofthe formulas;

 wherein: each m and mm is, independently, from 1 to 10; y is from 1 to10; E is N(R₁)(R₂) or N═C(R₁)(R₂); each R₁ and R₂ is, independently, H,a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, substituted orunsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is —C(═O)N(R₃)(R₄),—C(═O)R₃, or —C(═O)OR₃; provided that when T₄ is Bx-L, R₁ and R₂ are notboth H; or R₁ and R₂, together, are a nitrogen protecting group or arejoined in a ring structure that optionally includes an additionalheteroatom selected from N and O; and each R₃ and R₄ is, independently,H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₃ and R₄, together,are a nitrogen protecting group; or R₃ and R₄ are joined in a ringstructure that optionally includes an additional heteroatom selectedfrom N and O.
 2. The compound of claim 1 wherein one of T₁, T₂ or T₃ isL.
 3. The compound of claim 2 wherein T₃ is L.
 4. The compound of claim1 wherein T₄ is Bx—L.
 5. The compound of claim 1 wherein L is—O—(CH₂)₂—O—N(R₁)(R₂).
 6. The compound of claim 2 wherein R₁ is H orC₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl and R₂ is C₁-C₁₀ substitutedalkyl.
 7. The compound of claim 6 wherein R₁ is C₁-C₁₀ alkyl.
 8. Thecompound of claim 6 wherein R₂ is NH₃ ⁺ or N (R₃)(R₄) substituted C₁-C₁₀alkyl.
 9. The compound of claim 6 wherein R₁ and R₂ are both C₁-C₁₀substituted alkyl.
 10. The compound of claim 9 wherein the substituentson the C₁-C₁₀ substituted alkyls are, independently, NH₃ ⁺ or N(R₃)(R₄).11. The compound of claim 1 wherein Bx is adeninyl, guaninyl,hypoxanthinyl, uracilyl, thyminyl, cytosinyl, 2-aminoadeninyl or5-methylcytosinyl.
 12. The compound of claim 1 wherein R₁ and R₂ arejoined in a ring structure that can include at least one heteroatomselected from N and O.
 13. The compound of claim 12 wherein said ringstructure is imidazolyl, piperidinyl, morpholino or a substitutedpiperazinyl.
 14. The compound of claim 13 wherein said substitutedpiperazinyl is substituted with a C₁-C₁₂ alkyl.
 15. The compound ofclaim 1 wherein T₁ is a protected hydroxyl.
 16. The compound of claim 1wherein T₂ is an activated phosphorus-containing substituent group or aconnection to a solid support.
 17. The compound of claim 16 wherein saidsolid support material is microparticles.
 18. The compound of claim 16wherein said solid support material is controlled pore glass (CPG). 19.The compound of claim 4 wherein L is bound to an exocyclic aminofunctionality of Bx.
 20. The compound of claim 4 wherein L is bound to acyclic carbon atom of Bx.
 21. The compound of claim 4 wherein Bx isadeninyl, 2-aminoadeninyl or guaninyl.
 22. The compound of claim 4wherein Bx is a pyrimidine heterocyclic base and L is covalently boundto said base.
 23. The compound of claim 4 wherein Bx is a purineheterocyclic base and L is covalently bound to said base.
 24. Anoligomeric compound comprising a plurality of nucleoside units of thestructure:

wherein: T₄ of each nucleoside unit is, independently, Bx or Bx—L whereBx is a heterocyclic base moiety; one of T₅, T₆ and T₇ of eachnucleoside unit is, independently, L, hydroxyl, a protected hydroxyl, asugar substituent group, an activated phosphorus-containing substituentgroup, a connection to a solid support, a nucleoside, a nucleotide, anoligonucleoside or an oligonucleotide; another of T₅, T₆ and T₇ of eachnucleoside unit is, independently, a nucleoside, a nucleotide, anoligonucleoside or an oligonucleotide; the remaining one of T₅, T₆ andT₇ of each nucleoside unit is, independently, is L, hydrogen, hydroxyl,a protected hydroxyl, or a sugar substituent group; provided that on atleast one of said nucleoside units T₄ is Bx—L or at least one of T₅, T₆and T₇ is L; said group L having one of the formulas;

 wherein: each m and mm is, independently, from 1 to 10; y is from 1 to10; E is N(R₁)(R₂) or N═C(R₁)(R₂); each R₁ and R₂ is, independently, H,a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, substituted orunsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is —(C═O)N(R₃)(R₄),—C(═O)R₃, or —C(═O)OR₃, or R₁ and R₂, together, are a nitrogenprotecting group or are joined in a ring structure that optionallyincludes an additional heteroatom selected from N and O; and each R₃ andR₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, orR₃ and R₄, together, are a nitrogen protecting group or wherein R₃ andR₄ are joined in a ring structure that optionally includes an additionalheteroatom selected from N and O.
 25. The oligomeric compound of claim24 having 8 to 30 nucleoside units.
 26. The oligomeric compound of claim24 having 15 to 25 nucleoside units.
 27. The oligomeric compound ofclaim 24 wherein at least one of T₅, T₆ and T₇ is L.
 28. The oligomericcompound of claim 24 wherein at least one T₃ is L.
 29. The oligomericcompound of claim 24 wherein at least one T₄ is Bx—L.
 30. The oligomericcompound of claim 24 wherein L of one of said nucleoside units is—O—(CH₂)₂—O—N(R₁)(R₂).
 31. The oligomeric compound of claim 24 whereinR₁ is H, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl and R₂ is C₁-C₁₀substituted alkyl.
 32. The oligomeric compound of claim 31 wherein R₁ isC₁-C₁₀ alkyl.
 33. The oligomeric compound of claim 31 wherein R₂ is NH₃⁺ or N(R₃)(R₄) substituted C₁-C₁₀ alkyl.
 34. The oligomeric compound ofclaim 31 wherein R₁ and R₂ are both C₁-C₁₀ substituted alkyl.
 35. Theoligomeric compound of claim 34 wherein the substituents on the C₁-C₁₀substituted alkyls are, independently, NH₃ ⁺ or N(R₃)(R₄).
 36. Theoligomeric compound of claim 24 wherein Bx is adeninyl, guaninyl,hypoxanthinyl, uracilyl, thyminyl, cytosinyl, 2-aminoadeninyl or5-methylcytosinyl.
 37. The oligomeric compound of claim 24 wherein R₁and R₂ are joined in a ring structure that can include at least oneheteroatom selected from N and O.
 38. The oligomeric compound of claim37 wherein said ring structure is imidazolyl piperidinyl, morpholino ora substituted piperazinyl.
 39. The oligomeric compound of claim 38wherein said substituted piperazinyl is substituted with a C₁-C₁₂ alkyl.40. The oligomeric compound of claim 24 wherein T₅ is a protectedhydroxyl.
 41. The oligomeric compound of claim 24 wherein T₆ is anactivated phosphorus-containing substituent group or a connection to asolid support.
 42. The oligomeric compound of claim 41 wherein saidsolid support material is microparticles.
 43. The oligomeric compound ofclaim 41 wherein said solid support material is controlled pore glass(CPG).
 44. The oligomeric compound of claim 29 wherein L is bound to anexocyclic amino functionality of Bx.
 45. The oligomeric compound ofclaim 29 wherein L is bound to a cyclic carbon atom of Bx.
 46. Theoligomeric compound of claim 29 wherein Bx is adeninyl, 2-aminoadeninylor guaninyl.
 47. The oligomeric compound of claim 29 wherein Bx is apyrimidine heterocyclic base and L is covalently bound to said base. 48.The oligomeric compound of claim 29 wherein Bx is a purine heterocyclicbase and L is covalently bound to said base.
 49. The oligomeric compoundof claim 24 having 5 to 50 nucleoside units.
 50. An oligomeric compoundspecifically hybridizable with DNA or RNA comprising a sequence oflinked nucleoside units, wherein: said sequence is divided into a firstregion having linked nucleoside units and a second region being composedof linked nucleoside units having 2′-deoxy sugar moieties; said linkednucleoside units of at least one of said first or second regions areconnected by phosphorothioate linkages; at least one of said linkednucleoside units of said first region bearing a group L that iscovalently attached to the heterocyclic base or the 2′,3′ or 5′ positionof the sugar moiety; said group L having one of the formulas:

 wherein: each m and mm is, independently, from 1 to 10; y is from 1 to10; E is N(R₁)(R₂) or N═C(R₁)(R₂); each R₁ and R₂ is, independently, H,a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, substituted orunsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is —C(═O)N(R₃)(R₄),—C(═O)R₃, or —C(═O)OR₃; or R₁ and R₂, together, are a nitrogenprotecting group or are joined in a ring structure that optionallyincludes an additional heteroatom selected from N and O; and each R₃ andR₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, orR₃ and R₄, together, are a nitrogen protecting group; and or R₃ and R₄are joined in a ring structure that optionally includes an additionalheteroatom selected from N and O.
 51. The oligomeric compound of claim50 wherein Bx is adeninyl, guaninyl, hypoxanthinyl, uracilyl, thyminyl,cytosinyl, 2-aminoadeninyl or 5-methylcytosinyl.
 52. The oligomericcompound of claim 50 wherein L is —O—(CH₂)₂—O—N(R₁)(R₂).
 53. Theoligomeric compound of claim 50 wherein at least one of said linkednucleosides of said first region having said group L covalently attachedto the 2′, 3′ or 5′-position of the sugar moiety.
 54. The oligomericcompound of claim 53 wherein said group L is covalently attached to the2′-position of the sugar moiety.
 55. The oligomeric compound of claim 50wherein said nucleoside units of said first and second regions areconnected by phosphorothioate internucleoside linkages.
 56. Theoligomeric compound of claim 50 wherein said nucleoside units of saidfirst region are connected by phosphodiester internucleoside linkagesand said nucleoside units of said second region are connected byphosphorothioate internucleoside linkages.
 57. The oligomeric compoundof claim 50 wherein said nucleoside units of said first region areconnected by phosphorothioate internucleoside linkages and saidnucleoside units of said second region are connected by phosphodiesterinternucleoside linkages.
 58. The oligomeric compound of claim 50wherein said second region has at least three nucleoside units.
 59. Theoligomeric compound of claim 50 wherein said second region has at leastfive nucleoside units.
 60. The oligomeric compound of claim 50 having 5to 50 nucleoside units.
 61. The oligomeric compound of claim 50 having 8to 30 nucleoside units.
 62. The oligomeric compound of claim 50 having15 to 25 nucleoside units.
 63. The oligomeric compound of claim 50wherein at least one of said linked nucleosides of said first regionhaving said group L covalently attached to the heterocyclic base. 64.The oligomeric compound of claim 63 wherein L is bound to an exocyclicamino functionality of the heterocyclic base.
 65. The oligomericcompound of claim 63 wherein L is bound to a cyclic carbon atom of theheterocyclic base.
 66. The oligomeric compound of claim 63 wherein theheterocyclic base is adeninyl, 2-aminoadeninyl or guaninyl.
 67. Theoligomeric compound of claim 63 wherein the heterocyclic base is apyrimidine and L is covalently bound to said base.
 68. The oligomericcompound of claim 63 wherein the heterocyclic base is a purine and L iscovalently bound to said base.
 69. The oligomeric compound of claim 50wherein R₁ is H, C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl and R₂ isC₁-C₁₀ substituted alkyl.
 70. The oligomeric compound of claim 69wherein R₁ and R₂ are both C₁-C₁₀ substituted alkyl.
 71. The oligomericcompound of claim 70 wherein the substituents on the C₁-C₁₀ substitutedalkyls are, independently, NH₃ ⁺ or N(R₃)(R₄).
 72. The oligomericcompound of claim 69 wherein R₂ is NH₃ ⁺ or N(R₃)(R₄) substituted C₁-C₁₀alkyl.
 73. The oligomeric compound of claim 69 wherein R₁ is C₁-C₁₀alkyl.
 74. The oligomeric compound of claim 50 wherein R₁ and R₂ arejoined in a ring structure that can include at least one heteroatomselected from N and O.
 75. The oligomeric compound of claim 74 whereinsaid ring structure is imidazolyl, piperidinyl, morpholino or asubstituted piperazinyl.
 76. The oligomeric compound of claim 75 whereinsaid substituted piperazinyl is substituted with a C₁-C₁₂ alkyl.
 77. Theoligomeric compound of claim 50 further comprising a third region, saidthird region having substituted or unsubstituted 2′-O-alkyl nucleosideunits, said substituted 2′-O-alkyl nucleoside units bearing a group L,wherein said second region is positioned between said first and thirdregions.
 78. The oligomeric compound of claim 77 wherein said nucleosideunits of said first, second and third regions are connected byphosphorothioate linkages.
 79. The oligomeric compound of claim 77wherein said nucleoside units of said first and third regions areconnected by phosphodiester linkages and said nucleoside units of saidsecond region are connected by phosphorothioate linkages.
 80. Theoligomeric compound of claim 77 wherein said nucleoside units of saidfirst and third regions are connected by phosphorothioate linkages andsaid nucleoside units of said second region are connected byphosphodiester linkages.
 81. The oligomeric compound of claim 77 whereinsaid second region has at least three nucleoside units.
 82. Theoligomeric compound of claim 77 wherein said second region has at leastfive nucleoside units.
 83. The oligomeric compound of claim 77 whereinat least one of said 2′-O-alkyl nucleoside units of said third regionbears a 2′-aminooxy group having one of the formulas:

wherein: each m and mm is, independently, from 1 to 10; y is from 1 to10; E is N(R₁)(R₂) for N═C(R₁)(R₂); each R₁ and R₂ is, independently, H,a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl, substituted orunsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is —C(═O)N(R₃)(R₄),—C(═O)R₃, or —C(═O)OR₃; or R₁ and R₂, together, are a nitrogenprotecting group or are joined in a ring structure that optionallyincludes an additional heteroatom selected from N and O; and each R₃ andR₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, orR₃ and R₄, together, are a nitrogen protecting group; and or R₃ and R₄are joined in a ring structure that optionally includes and additionalheteroatom selected from N and O.